Methods for Neural Conversion of Human Embryonic Stem Cells

ABSTRACT

The present invention relates generally to the field of cell biology of stem cells, more specifically the directed differentiation of pluripotent or multipotent stem cells, including human embryonic stem cells (hESC), somatic stem cells, and induced human pluripotent stem cells (hiPSC) using novel culture conditions. Specifically, methods are provided for obtaining neural tissue, floor plate cells, and placode including induction of neural plate development in hESCs for obtaining midbrain dopamine (DA) neurons, motorneurons, and sensory neurons. Further, neural plate tissue obtained using methods of the present inventions are contemplated for use in co-cultures with other tissues as inducers for shifting differentiation pathways, i.e. patterning.

FIELD OF THE INVENTION

The present invention relates generally to the field of cell biology ofstem cells, more specifically the directed differentiation ofpluripotent or multipotent stem cells, including human embryonic stemcells (hESC), somatic stem cells, and induced human pluripotent stemcells (hiPSC) using novel culture conditions. Specifically, methods areprovided for obtaining neural tissue, floor plate cells, and placodeincluding induction of neural plate development in hESCs for obtainingmidbrain dopamine (DA) neurons, motoneurons, and sensory neurons.Further, neural plate tissue obtained using methods of the presentinventions are contemplated for use in co-cultures with other tissues asinducers for shifting differentiation pathways, i.e. patterning.

BACKGROUND OF THE INVENTION

The differentiation capacity of embryonic and somatic stem cells haveopened possibilities for cell replacement therapies for genetic,malignant, and degenerative diseases. Neurodegenerative disorders,conditions, and diseases, and their treatment by cell-based therapiesrepresent a promising means of preventing, reducing or eliminating thesymptoms. Such disorders include Huntington's disease, Alzheimer's,Parkinson's, and amyotrophic lateral sclerosis. They also provide asource of cells for screening for critical small molecules (i) thatcould be useful in for treatment of disease; or (ii) for determining thecell fate of neural tissue. Further, these cells were studied in orderto characterize key genes, mRNA transcripts, and proteins relevant innormal or pathological lineages.

Neural development is dictated in time and space by a complex set ofsignals that instruct neural precursor identity. While significantprogress was made in animal models, human neural development remainsmuch less understood.

Previous studies reported directed differentiation of mouse (Wichterleet al., 2002; Barbed. et al., 2003; Watanabe et al., 2005) and human(Perrier et al., 2004; Li et al., 2008; Eiraku et al., 2008) ESCs intospecific neuron types in response to patterning factors defininganterior/posterior (A/P) and dorso-/ventral (D/V) CNS identity. Thesestudies demonstrate evolutionary conservation of signaling systems thatspecify the major CNS regions. In mammals, sonic hedgehog (SHH) is thekey ventralizing factor acting in a dose-dependent manner to specify thevarious ventral cell types including cells expressing floor plate (FP)in primary neural explants (Briscoe and Ericson, 1999) and in mouse EScells (Mizuseki et al., 2003). While application of SHH to hESC-derivedneural cells was shown to induce various ventral neuron types, thederivation of floor plate (FP) tissue itself was not reported. As FP isone of the most important signaling centers for inducing differentiationpathways and subsequent committed cell lineage, the ability to produceFP from human ES cells would be a major step forward in furtheringstudies of early human neural development. Furthermore, little is knownabout FP development in humans, due to lack of accessibility to tissue.

In animals, the FP is a major site of SHH production and several humandevelopmental disorders are related to alterations in midline SHHsignaling (Mullor et al., 2002) including certain forms ofholoprosencephaly and microphthalmia, skeletal disorders includingvarious cleft plate syndromes, and tumor conditions such as Gorlin'ssyndrome; a rare genetic disorder caused by a mutation in the SHHreceptor Patched 1. However it is not known whether similar alterationsin midline SHH signaling would induce these diseases in humans.

Therefore there is a critical need for inducing human floor plate tissuefrom human embryonic stem cells (hESCs) for providing a source of humanfloor plate cells. These human floor plate cells are necessary for usein medical research for determining causes and treatments ofdevelopmental diseases in humans and for comparative developmentalstudies of human neural patterning and axonal pathfinding.

SUMMARY OF THE INVENTION

The present invention relates generally to the field of cell biology ofstem cells, more specifically the directed differentiation ofpluripotent or multipotent stem cells, including human embryonic stemcells (hESC), somatic stem cells, and induced human pluripotent stemcells (hiPSC) using novel culture conditions. Specifically, methods areprovided for obtaining neural tissue, floor plate cells, and placodeincluding induction of neural plate development in hESCs for obtainingmidbrain dopamine (DA) neurons, motorneurons, and sensory neurons.Further, neural plate tissue obtained using methods of the presentinventions are contemplated for use in co-cultures with other tissues asinducers for shifting differentiation pathways, i.e. patterning.

The present invention is related to methods of obtaining populations ofneural progenitor cells derived from human embryonic stem cells (hESCs),in particular for obtaining neural plate tissue. Specifically, methodsof the present inventions induce neural plate floor development in hESCsfor obtaining dopamine (DA) nerve cells. Further, neural plate floortissue obtained using methods of the present inventions are contemplatedfor use in co-cultures with other tissues as inducers for shiftingdifferentiation pathways, i.e. patterning.

The present invention relates generally to the field of cell biology ofstem cells, more specifically the directed differentiation ofpluripotent or multipotent stem cells, including human embryonic stemcells (hESC), somatic stem cells, and induced human pluripotent stemcells (hiPSC) using novel culture conditions.

The present inventions provide a method of producing a human neural cell(neural stem cells, neuronal subtypes, mature neurons, cells of a neurallineage) by (i) obtaining stem cells (hESCs, hiPSCs, somatic stem cells,cancer stem cells, human or mammalian pluripotent or multipotent cells);and (ii) culturing the human stem cell under conditions that block SMADsignaling. In a preferred embodiment, the methods for culture includeconditions in a feeder-free system. In a preferred embodiment, the stemcells are cultured in a monolayer. A preferred embodiment contemplatedthe use of media that is supplemented with compounds Noggin and/orDorsomorphin and SB431542.

In one embodiment the inventions provide a kit comprising a firstinhibitor of Small Mothers Against Decapentaplegic (SMAD) proteinsignaling and a second inhibitor of Small Mothers AgainstDecapentaplegic (SMAD) protein signaling. In one embodiment, said firstinhibitor is selected from the group consisting of a disulfide-linkedhomodimer of Noggin (SEQ ID NO:50), Dorsomorphin, LDN-193189,combination thereof and mixture thereof. In one embodiment, said Nogginis selected from mouse, human, rat, and xenopus. In one embodiment, saidis Noggin is (SEQ ID NO:50) In one embodiment, said second inhibitorinhibits an anaplastic lymphoma kinase signaling pathway. In oneembodiment, said second inhibitor inhibits a signaling pathway selectedfrom the group consisting of Lefty, Activin, and TGFbeta. In oneembodiment, said second inhibitor inhibits both activins and nodalsignaling. In one embodiment, said second inhibitor is4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide(SB431542) and derivatives thereof. In one embodiment, said kit furthercomprises a human stem cell. In one embodiment, the kit furthercomprises instructions.

In one embodiment the inventions provide a composition comprising a stemcell, a first inhibitor of Small Mothers Against Decapentaplegic (SMAD)protein signaling and a second inhibitor of Small Mothers AgainstDecapentaplegic (SMAD) protein signaling. In one embodiment, said firstinhibitor is selected from the group consisting of a disulfide-linkedhomodimer of Noggin, Dorsomorphin, LDN-193189, combination thereof andmixture thereof. In one embodiment, said is Noggin is selected frommouse, human, rat, and xenopus. In one embodiment, said is Noggin is(SEQ ID NO:50) In one embodiment, said second inhibitor inhibits theLefty/Activin/TGFbeta pathways by blocking phosphorylation of the ALK4,ALK5 and ALK7 receptors. In one embodiment, said second inhibitorinhibits activin/nodal signaling. In one embodiment, said secondinhibitor is4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide(SB431542) and derivatives thereof. In one embodiment, said stem cell isselected from the group consisting of human embryonic stem cells (hESC),somatic stem cells, and induced human pluripotent stem cells (hiPSC).

In one embodiment the inventions provide a method for inducingdifferentiation in stem cell, comprising, a) providing: i) a cellculture comprising human stem cells, ii) a first inhibitor of SmallMothers Against Decapentaplegic (SMAD) protein signaling, iii) a secondinhibitor of Small Mothers Against Decapentaplegic (SMAD) proteinsignaling, and b) contacting said stem cells with said first inhibitorof Small Mothers Against Decapentaplegic (SMAD) protein signaling andsaid test compound under conditions for inducing differentiation in astem cell into a non-default differentiated cell. In one embodiment,said first inhibitor is selected from the group consisting of adisulfide-linked homodimer of Noggin, Dorsomorphin, LDN-193189,combination thereof and mixture thereof. In one embodiment, said isNoggin is selected from mouse, human, rat, and xenopus. In oneembodiment, said is Noggin is (SEQ ID NO:50) In one embodiment, saidsecond inhibitor is a ALK4 receptor inhibitor. In one embodiment, saidsecond inhibitor is4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide(SB431542) and derivatives thereof. In one embodiment, said non-defaultdifferentiated cell is a neural progenitor cell. In one embodiment, saidnon-default differentiated cell is a part of a population of culturedcells. In one embodiment, said non-default differentiated cell is atleast 10% up to 100% of said population of cultured cells. In oneembodiment, said non-default differentiated cell in a population ofcultured cells expresses paired box gene 6 protein. In one embodiment,said paired box gene 6 protein is expressed in at least 10% of saidpopulation of cultured cells. In one embodiment, said stem cell isselected from the group consisting of human embryonic stem cells (hESC),human somatic stem cells, and induced human pluripotent stem cells(hiPSC). In one embodiment, said non-default differentiated cell is aneural cell. In one embodiment, said neural cell is selected from thegroup consisting of dopamine positive neurons and floor plate cells.

In one embodiment the inventions provide a composition comprisingisolated human embryonic neural cells. In one embodiment, said isolatedhuman embryonic neural cells were derived from human embryonic cells. Inone embodiment, said human embryonic neural cells are cultured in vitro.In one embodiment, said human embryonic neural cells are attached cells.In one embodiment, said composition is a co-culture further comprising asecond cell type.

In one embodiment the inventions provide a method for screeningbiological agents, comprising, a) providing: i) a cell culturecomprising human embryonic stem cells (hESCs), and ii) a test compound,and b) contacting said stem cells with said test compound underconditions for inducing neural floor plate cells. In one embodiment,said test compound is sonic hedgehog or fragment thereof. In oneembodiment, said human embryonic stem cells are rosette-stage neuralcells.

In one embodiment the inventions provide a method for providingdifferentiated cells, comprising, a) providing i) a cell culture ofhuman embryonic stem cells (hESCs), and ii) a compound for inducingdifferentiation, and b) contacting said stem cells with said testcompound under conditions for inducing neural floor plate cells. In oneembodiment, said compound is sonic hedgehog protein or fragment thereof.In one embodiment, said inducing consists of increasing a characteristicselected from the group consisting of flat cellular morphology,expressing sonic hedgehog, expressing forkhead box protein A2 (FOXA2),expressing Netrin-1, and expressing F-Spondin compared to saidcharacteristic expressed in said human embryonic stems cells culturedwithout said test compound. In one embodiment, said inducing consists ofdecreasing a characteristic selected from the group consisting ofrosette structures, BF1 expression, paired box homeotic gene-6 (PAX6)expression, NK6 homeobox 1 (NKX6.1), homeobox protein SIX6 expressioncompared to said characteristic in said human embryonic stems cellscultured without said test compound. In one embodiment, the methodfurther provides and comprises a Noggin protein and an agent forblocking phosphorylation of a receptor selected from the groupconsisting of activin receptor-like kinase 4 (ALK4), activinreceptor-like kinase 5 (ALK5) and activin receptor-like kinase 7 (ALK7)receptors and contacting said human stem cells with said noggin and saidagent to human stem cells before adding said compound. In oneembodiment, the method further provides an antibody, wherein saidantibody is dickkopf homolog 1 (DKK-1) antibody, and contacting saidstem cells with said antibody for reducing DKK-1 protein function. Inone embodiment, the method further provides and comprises a caudalizingfactor selected from the group consisting of wingless-type MMTVintegration site family, member 1 (Wnt-1), and Retinoic Acid (RA). Inone embodiment, the method further provides and comprises a neuroninducing compound and step c) adding said neuron inducing compound forinducing progenitor neurons. In one embodiment, said dopamine neuronsexpress a marker selected from the group consisting of corin, serinepeptidase (CORIN) and nephroblastoma overexpressed gene (NOV). In oneembodiment, said progenitor neurons are dopamine neurons express amarker selected from the group consisting of LIM homeobox transcriptionfactor 1, beta (LMX1B) and neurogenin 2 (NGN2). In one embodiment, themethod further provides and comprises a stem cell, and step d)co-culturing said human neural floor plate cells with said stem cellsfor producing neurite outgrowth from said stem cells.

In one embodiment the inventions provide a neural floor plate cellproduced by the methods described herein. In one embodiment theinventions provide a placode cell produced by the methods describedherein. In one embodiment the inventions provide a lens cell produced bythe methods described herein.

The invention contemplates methods for assessing the neural identity ofthe derived neural cells. This method may be through morphologicalmeans, functional assessment, and measurement of expression ordownregulation of proteins associated with certain lineages. In apreferred method, dopaminergic activity or functional assays for motorneurons are utilized.

The present method can by employed to deliver agents or neural cells tothe brain in an effective amount for diagnosis, prevention, treatment ofdisease, disorders, or for patients suffering from nerve damage formstroke. Such cells were co-committed towards a neural fate.

In one embodiment, the present invention contemplates a compositioncomprising isolated human embryonic floor plate cells. In oneembodiment, the isolated human embryonic floor plate cells were derivedfrom human embryonic cells. In one embodiment, the human embryonic floorplate cells are cultured in vitro. In one embodiment, the humanembryonic floor plate cells are attached cells. In one embodiment, thecomposition is a co-culture further comprising a second cell type.

In one embodiment, the present invention contemplates a method forscreening biological agents, comprising, a) providing: i) a cell culturecomprising human embryonic stem cells (hESCs), and ii) a test compound,and b) contacting said stem cells with said test compound underconditions for inducing neural floor plate cells. In one embodiment, thetest compound is sonic hedgehog or fragment thereof. In one embodiment,the human embryonic stem cells are rosette-stage neural cells.

In one embodiment, the present invention contemplates a method forproviding differentiated cells, comprising, a) providing: i) a cellculture of human embryonic stem cells (hESCs), and ii) a compound forinducing differentiation, and b) contacting said stem cells with saidtest compound under conditions for inducing neural floor plate cells. Inone embodiment, the compound is sonic hedgehog protein or fragmentthereof. In one embodiment, the inducing consists of increasing acharacteristic selected from the group consisting of flat cellularmorphology, expressing sonic hedgehog, expressing forkhead box proteinA2 (FOXA2), expressing Netrin-1, and expressing F-Spondin compared tosaid characteristic expressed in said human embryonic stems cellscultured without said test compound. In one embodiment, the inducingconsists of decreasing a characteristic selected from the groupconsisting of rosette structures, BF1 expression, paired box homeoticgene-6 (PAX6) expression, NK6 homeobox 1 (NKX6.1), homeobox protein SIX6expression compared to said characteristic in said human embryonic stemscells cultured without said test compound. In one embodiment, the methodfurther provides Noggin protein and an agent for blockingphosphorylation of a receptor selected from the group consisting ofactivin receptor-like kinase 4 (ALK4), activin receptor-like kinase 5(ALK5) and activin receptor-like kinase 7 (ALK7) receptors andcontacting said human stem cells with said noggin and said agent tohuman stem cells before adding said compound. In one embodiment, themethod further provides an antibody, wherein said antibody is dickkopfhomolog 1 (DKK-1) antibody, and contacting said stem cells with saidantibody for reducing DKK-1 protein function. In one embodiment, themethod further provides a caudalizing factor selected from the groupconsisting of wingless-type MMTV integration site family, member 1(Wnt-1), and Retinoic Acid (RA). In one embodiment, the furthercomprises, providing, a neuron inducing compound and step c) adding saidneuron inducing compound for inducing progenitor neurons. In oneembodiment, the dopamine neurons express a marker selected from thegroup consisting of corin, serine peptidase (CORIN) and nephroblastomaoverexpressed gene (NOV). In one embodiment, the progenitor neurons aredopamine neurons express a marker selected from the group consisting ofLIM homeobox transcription factor 1, beta (LMX1B) and neurogenin 2(NGN2). In one embodiment, the method further comprises, providing, stemcells, and step d) co-culturing said human neural floor plate cells withsaid stem cells for producing neurite outgrowth from said stem cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 showing exemplary dual SMAD inhibition that allowed for a highlyefficient feeder-free neural induction in adherent cultures within sevendays. (a) Differentiation scheme used for achieving neural inductionwere achieved with the combination of SB431542, an ALK inhibitor, andNoggin, a BMP inhibitor. (b) The dual SMAD inhibition greatly improvesneural differentiation (PAX6 expression, green) to greater than 80%.Infrequent neural differentiation (<10% PAX6⁺ cells) were observed whenthe single factors are used. (c) Real-Time PCR for early germ layermarkers CDX2, SOX1, SOX17 and Brachyury. (d) Immunoflouresence for OCT4(red) and PAX6 (green) expression indicates rapid neutralization occursby day 7. (e) Real-Time PCR for PAX6, OTX2, FGF5, OCT4 during dual SMADinhibition reveals an epistem cell intermediate at day 5. (f) Real-TimePCR for neural and neuronal markers during dual SMAD inhibitiondifferentiation towards neuroectoderm. (g) A BAC reporter line(HES5-GFP) was used to quantify the percentage of neural induction forthe method using MS5 stromal cells (with Noggin) or dual SMAD inhibition(SB431542 and Noggin). Error bars represent S.E.M. and the p-value wasdetermined using Student's T-test. Abbreviations: N, Noggin; SB,SB431542; KSR, knock-out serum replacement medium; N2, N2 medium. Scalebars: (b)—200 μm; (d)—50 μm.

FIG. 2 showing exemplary nuclear localization of SMAD4 diminishes whenhESC cells are treated with Noggin and SB43152 for 24 hours. Aproportion of SMAD4 redistributes to a perinuclear localizationresulting in a less defined cytoplasmic-to-nuclear border.

FIG. 3 showing an exemplary model of proposed mechanisms that contributeto the action of Noggin and SB431542. These include destabilizing theTGF/activin- and Nanog-mediated pluripotency network, suppression ofmesendodermal fates by inhibiting endogenous activin and nodal signals,and promoting neuralization of primitive ectoderm through BMPinhibition. (a) At high density, primarily CNS cells that are PAX6+ areformed, which are capable of giving rise to R-NS cells and patternableneuronal populations of motoneurons and dopaminergic neurons within 19 dof differentiation. (b) At lower densities, both CNS fates with theproperties described in (a) and neural crest fates are observed. Neuralcrest lineages include melanocytes and neural crest precursor cellsamenable to patterning and subtype specification responses. In additionto cell density, it is likely that further manipulation of signalingpathways, including BMP pathways, will skew that ratio of CNS versusneural crest fates. Solid arrows indicate demonstrated cell fatepotential; dashed arrows indicate proposed cell fates on the basis ofcurrent literature.

FIG. 4 showing exemplary nanog Real-Time gene expression. hESC treatedwith knock-out serum (KSR), Noggin (N), SB431542 (SB), or Noggin andSB431542 (N+SM) in KSR were examined for Nanog expression. The mostdramatic downregulation was observed with the addition of SB431542. Theerror bars represent S.E.M.

FIG. 5 showing exemplary GFP expression of HES5-GFP BAC reporter hESCGFP were observed under both conditions for neural induction (Noggin onMS5s or Noggin with SB431542 at day 13 of differentiation.

FIG. 6 showing exemplary endoglin (CD105) expression on MS5 feedercells. MS5 cells used to differentiate hESC are uniformly positive forEndoglin (CD105) expression based on FACS analysis compared to hESCdifferentiated on day 13 using combined SMAD suppression. Endoglinexpression was used to discriminate and remover MS5 cells from HES5-BAChESC.

FIG. 7 showing exemplary neuralization of hESC by dual SMAD inhibitionpermits a pre-rosette, neural stem cell with dopaminergic and motorneuronal potential. The PAX6 positive neural tissue (green) expressedrosette markers (red) (a) Nestin, (b) PLZF, (c) ZO1. (d) Rosettes areformed when PAX6⁺ tissue is passaged to conditions promoting rosettes(BASF) continued by KI67 (green) and luminal phospho-Histone H3 (red)expression, evidence of interkinetic nuclear migration. In the absenceof factors that confer regional neuronal specificity, the PAX6⁺ neuraltissue (green) expressed (e) OTX2, and (f) BF1, indicating that thetissue defaults to forebrain specification. Neural crest could beidentified on the periphery of the PAX6 positive tissue (green) based on(g) AP2, (h) HNK1, (i) PAX7, and (j) p75 expression (red). Upon passage,the neural crest cells gave rise to (k) pigmented cells (l) thatexpressed HMB45 (green), indicating melanosome synthesis. (m)Dopaminergic neuronal patterning was initiated with the addition ofsuper sonic on day 5-9, followed by the addition of BDNF, ascorbic acid,sonic hedgehog, and FGF8 on day 9-12. Dopaminergic cells were maturatedon days 12-19 with BDNF, ascorbic acid, GDNF, TGFb3, and cAMP. Motorneuronal patterning was initiated at day 5 with the addition of BDNF,ascorbic acid, sonic hedgehog, and retinoic acid. Cells were passaged onday 11. (n-p) Without passage, tyrosine hydroxylase (TH) positive cellscould be observed by day 19. (p) When passaged en bloc on day 12, moremature processes from TH positive cells were observed. For motoneuroninduction, nuclear expression of the motor neuron markers (q) ISL1 and(r) HB9 were observed within a total of 19 days of differentiation fromhESC. Scale bars: (a, b, c, e, f, g, h, i, j, o, p, q, and r)—100 μm;(c,d)—50 μm; (k,l,n)—200 μm.

FIG. 8 showing exemplary plating density influences PNS vs. CNS cellgeneration. Initial hESC plating density determines the ratio ofneural-crest (HNK1, p75; red) to neural tissue (PAX6; green) present atday 11 of differentiation, with higher densities favoring neuraldifferentiation.

FIG. 9 showing exemplary induced pluripotent stem cells (IPS) weredifferentiated to neural tissue using dual SMAD inhibition and arepatternable to dopaminergic neurons and motor neurons. (a-i, ii) Two IPSclones (IPS^(C14), IPS^(C27)) were generated and screened for OCT4 (red)as well as additional pluripotency factors (Tra-1-81, Tra-1-60, SSEA-4and Nanog). (b-i,ii) the two clones were neutralized by dual SMADinhibition (PAX6 expression, green), and neural crest could be observedby HNK1 staining (c-i,ii). Neural tissue from the IPS clones could beinduced to form rosette-NSCs (d-i,ii) based on KI-67 (red) andphospho-histone H3 (green) expression, motor neurons (e-i,ii) based onHB9 expression (green), and dopaminergic neurons (f-i,ii) based on TUJ1(green) and TH (red) co-expression. Scale bars: 200 μm—(a); 50μm—(b,c,d,e,f).

FIG. 10 showing exemplary combined SMAD inhibition during the first 5days of neural-induction. Homogeneous PAX6 expression was observed onday 11 when SB431542 and Noggin, supplemented in the media, werewithdrawn on day 5.

FIG. 11 showing exemplary derivation of Six1+ placodal precursors usinga modified N-SB protocol. A) Schematic illustration of timed nogginwithdrawal paradigm to determine temporal requirement for endogenous BMPsignaling in placode cell specification. B) Relative induction ofplacodal markers (Dlx3, Eya1, and Six1) comparing modified N-SB protocolas described in (A) to N-SB treatment maintained throughoutdifferentiation (SBN condition). Optimal co-expression of Dlx3, Eya1,and Six1 was observed when the cells are treated with noggin for 48hours. Data represent fold changes of mRNA expression measured byqRT-PCR at day 11. C) Immunocytochemical analyses showing Six1 (placodalmarker) and Pax6 (anterior neuroectoderm marker) expression at day 11 ofdifferentiation. Cells treated with the modified N-SB protocol (nogginwithdrawal after 2 days of differentiation) show high percentages ofSix1+ cells. D) Approximately seventy percent of cells generated usingmodified N-SB conditions (2 days of noggin) are Six1+ compared tostandard (anterior neurectoderm-inducing) 11 days of noggin treatment.

FIG. 12 showing exemplary temporal global gene expression profilesduring human ES cell derived placode specification. A-D) Pair-wisecomparison at day 5, day 7, day 9 and day 11 of differentiation of mostthe differentially expressed genes in hESC progeny subjected to themodified (placode-inducing) versus standard (anteriorneuroectoderm-inducing) N-SB protocol. E) Unsupervised clustering ofmicroarray data segregates data according to replicates, temporalsampling and treatment conditions. F) Principal component analysis ofdata confirms close temporal correlation of samples during human ES celldifferentiation with increasing separation of modified versus standardN-SB treated cells at later differentiation stages

FIG. 13 showing exemplary derivation of hESC placode derived sensoryneurons. A-C) Immunocytochemical analysis at day 20 of differentiationdemonstrates that placodal precursor cells efficiently yield neuronsthat initially retain Six1 expression. D, E) Sensory neuron identity isconfirmed by expression of Brn3A and Isl1 in the majority of neuronsderived from Six1+ clusters. F) At day 40 differentiation neurons showincreased expression of peripherin and decreased levels of Tuj1 stainingcharacteristic of a mature peripheral neuron fate. G) Schematicillustration of marker expression during sensory neuron specificationfrom hESC derived placodal cells.

FIG. 14 showing exemplary prospective isolation of hESC derived placodalprecursors. A) At day 11 of differentiation hESC derived cells aresegregated into mutually exclusive p75+ and a Forse1+ precursor celldomains. B) FACS analysis at day 11 of differentiation for expression ofp75 and HNK1. C) qRT-PCR data for Six1 mRNA expression followingseparation of cells based on the expression of p75 and HNK1. Cellssingle positive for p75 but negative for HNK1 (prospective placodalprecursors) showed a dramatic increase in Six1 mRNA expression comparedto other groups. D) An increase in the fraction of cells that arepositive for p75 and negative for HNK1 is observed when precursors arederived under modified (placode-inducing) compared to the standard(anterior neuroectoderm-inducing) N-SB induction conditions.

FIG. 15 showing exemplary high SHH levels increase FOXA2 and decreaseBF1 expression. (A) Passage 1, Day 21 of neural differentiation shows noeffect of SHH treatment when added at Day 15. Results quantified onright, *p<0.01 N=3. Scale bar, 200 um. (B) Day 21 of neuraldifferentiation shows a reduction of rosette like structures after SonicC25II treatment Day 9. Loss of rosettes quantified on right, *p<0.01N=4. Scale bar, 100 um. (C) Sonic C25II treatment results in a decreaseof BF1 and an increase in Foxa2 at Day 21. Quantified on right, *p<0.05N=4. Scale bar, 200 um. (D) Day 21 of neural differentiation reveals adecrease in ZO1/BF1+ rosette structures. This decrease is quantified,*p<0.01 N=4. Scale bar, 50 um. (E) Decrease in PAX6 expression at Day 21after Sonic C25II treatment. This decrease is quantified, *p<0.01 N=4.Scale bar, 200 um. (F) Dose response curve comparing Sonic and SonicC25II efficacy on FOXA2 induction. (E) Dose response curve of SonicC25II comparing the induction of FP markers (FOXA2 and Netrin-1) toanother SHH responsive gene NKX6.1.

FIG. 16 showing exemplary floor plate induction has an early, shorttemporal patterning window (A) Schematic showing different time pointsof Sonic C25II additions during neural induction protocol. (B-C) Headingon the left delineates the day Sonic C25II was added, heading on the topdelineates when the assay was stopped. The earlier Sonic C25II is added,and the longer the cells are exposed to it, leads to very highpercentages of FOXA2. (C) This result is quantified, *p<0.01 N=3. Scalebars, 200 um, high magnification, 50 um. (D) Extended treatment withSonic C25II (9 days of exposure) does not yield increased FOXA2induction. (E) Schematic of optimal protocol for FOXA2 induction to beused for the rest of the study.

FIG. 17 showing exemplary hESC derived FP is functional (A) Schematicshowing when conditioned media was collected. (B) ELISA showing anincrease in levels of Netrin-1 secreted into the media at Days 9 and 11when Sonic C25II is added early to the neural induction, *p<0.01 N=3.(C) Conditioned media from NSB and NSB+Sonic C25II was collected andplaced on cultures containing NSB derived neural precursor cells qRT-PCRshowing an induction of ventral genes (NKX6.1 and NKX2.1) as well as theSHH responsive gene (GLI2). These inductions are repressed in thepresence of the SHH antagonist cyclopamine. (C′) The induction of NKX6.1is shown at the level of the protein using a GFP expressing line.*p<0.01 compared to NSB CM, #p<0.05 compared to FP CM, N=3. Scale bar,200 um. (D and E) Neural explants isolated from E8.5 neurectodermco-cultured with NSB+Sonic C25II tissue show ectopic FOXA2 staining.Inset shows co-localization of M6 (Green) and FOXA2 (Red). (E) This datais quantified, *p<0.001 N=4 explants. Scale bar, 50 um.

FIG. 18 showing exemplary transcriptional analysis that revealed novelgenes involved in FP development. (A-J) qRT-PCR data showing time courseof expression over the length of the 11 day protocol. The genes lookedat represented different populations including FP markers (A-D), SHHresponsive genes (E-G), neural markers (H), AN markers (I and J), andgenes involved in mesodermal and endodermal commitment (K and L). (M-R)Detailed time course microarray analysis (M-N) GO terms for Day 7 (M)and Day 11 (N) showing increase or decrease compared to NSB control. FPcondition shows enrichment in genes associated with axon guidance andsecreted proteins, while showing a decrease in genes associated withanterior neurectoderm development. (O-R) Pair wise comparisons showinggenes up and down regulated compared to NSB control condition at Day 3(O), Day 5 (P), Day 7 (Q), and Day 11 (R).

FIG. 19 showing exemplary DKK-1 inhibition of FP induction. (A) qPCR forDKK-1 expression in control NSB condition over time. (B) ELISA measuringDKK-1 protein levels in the media at Day 5, 7, and 11 showing a decreasein Dkk-1 levels after Sonic C25II treatment, *p<0.05 N=3. (C) qPCR forDKK-1 expression in NSB+Sonic C25II condition over time. (D and E) qPCRfor BF1 (D) and FOXA2 (E) showing an increase in BF1 and decrease ofFOXA2 after DKK-1 addition, and an increase in FOXA2 when DKK-1 antibodyis added. (F) Immunostaining for FOXA2 showing a decrease in FOXA2+cells when DKK-1 is added. Scale bar, 200 um. (G) qPCR for BF1expression showing that DKK-1 antibody treatment leads to a decreasedexpression at earlier time points (Day 3-Day 5). (H and I) Earlyaddition of DKK-1 antibody leads to an increase of FOXA2 expression, buthas no effect when added at later timepoints. (I) Immunocytochemicaldata demonstrating that Dkk-1 treatment starting at day 5 ofdifferentiation (or later) does not enhance SHH-mediated FOXA2expression. (J-K″) hESC transduced with either control or BF1 shRNA (Jand K), GFP is a marker of transduction (A′ and B′). When differentiatedto neural tissue, a reduction of BF1 is seen at the level of the proteincompared to control (J″ and K″). Scale bars, A and B 100 um, J″ and K″200 um. (L) qRT-PCR analysis at Day 11 showed an increase in FP markers(FOXA2, SHH, Netrin-1 and F-Spondin) in the BF1 shRNA line compared tothe control, p<0.01 (M) BF1 shRNA leads to an upregulation of FOXA2 seenat the level of the protein. Scale bar, 200 um.

FIG. 20 showing exemplary hESC derived FP was shifted along the A/P axis(A) Immunostaining reveals an increase in FOXA2 in response to FGF8,Wnt-1, and Retinoic Acid. Scale bar, 200 um. (B) qPCR showingcaudilizing agents such as FGF8, Wnt-1, and Retinoic Acid (RA) lead toan increase in FOXA2 and a reduction in SIX6 compared to NSB+SonicC25II. (C) qPCR for a panel of midbrain FP markers (CORN and NOV) andmidbrain DA progenitor markers (LMX1B, NGN2, and EN1). In particular,Writ-1 treatment causes an upregulation of both midbrain FP markers aswell as midbrain DA progenitor markers. (D) FP cells were transfectedwith Shh enhancer that drives expression to the anterior ventral axis(SBE2) or midbrain ventral axis (SBE1). The default FP exhibits SBE2activity indicating an anterior location. This is abolished upon Wnt1and FGF8 addition and SBE1 activity is now seen suggesting a shift fromanterior identity to midbrain. Scale bar, 200 um. (E) Schematic of FPversus AN specification during hESC differentiation. Neuraldifferentiation is initiated upon exposure to Noggin and SB431542. SHEexposure, starting at day 1 of differentiation, induces FPdifferentiation and via inhibition of DKK-1 and BF1 suppresses ANspecification. The regional identity of the resulting FP cells isanterior by default but posterior FP tissue were induced in the presenceof caudalizing factors such as Wnt-1, FGFF8 or RA.

FIG. 21 showing exemplary hESC derived FP expresses the appropriatemarkers (A) qRT-PCR data at Day 11 showing an increase in floor platemarkers FOXA2, SHH, Netrin-1, and F-Spondin relative to control NSBconditions. (B) Table quantifying results of immunostaining experiments.(C-F) Immunostaining of FOXA2+ cells reveals co-labelling with fewmarkers such as (C) Nestin, (D) SOX2, (E) Nkx2.2, and (F) Tuj1. Scalebar, (D and F, 50 um) (E and G, 100 um). (G-H) qRT-PCR data at Day 11showing levels of FOXA2 and SOX17 cells differentiated with NSB+SonicC25II treatment and cells differentiated towards an endodermal lineage.SOX17 is not expressed in Sonic C25II conditions but is highly expressedin the endoderm. This is shown at the level of the protein byimmunostaining (H). Scale bar, 200 um.

FIG. 22 showing exemplary co-culture of cerebellar plate explants on FPcells induces neurite outgrowth. Cerebellar explants from E8.5 mousewere plated on NSB neural cells or NSB+Sonic (FP) cells. After 3 daysconsiderable neurite outgrowth was observed in the NSB+Sonic (FP)condition compared to control.

FIG. 23 showing exemplary qPCR validates genes changing in microarray.(A) qPCR for FP genes showing an enrichment in Sonic C25II conditioncompared to NSB control. (B-D) qPCR validating novel genes that changesin the Sonic C25II condition compared to NSB control condition.

FIG. 24 showing exemplary BF1 expression inhibits FP induction (A)qRT-PCR at two points during neural differentiation showing a decreasein BF1 levels in the BF shRNA hESC line compared to control, *p<0.01N=3. (B) Cell cycle analysis revealed no differences in the cell cyclekinetics of the two lines. (C) hESC expressing BF1 visualized by GFP.Scale bar, 20 um. (D) Cells overexpressing BF lack FOXA2+ expression.Scale bar, 200 um. (E) qRT-PCR data at Day 11 showing a lack of FPinduction in BF1 expressing hESC after Sonic C25II treatment.

FIG. 25 showing exemplary early WNT1 addition along FP differentiationcan cause DA neuron differentiation (A) Adding WNTs or GSK3β-Inhibitor(BIO 100 nM) early can increase FOXA2 expression. (B) Addition of WNT1to later stage neural rosette cells has no effect on FOXA2 induction,scale bar 200 um. (C) WNT1 treated FP cultures can give rise to DANeurons expressing FOXA2, scale bar 50 um.

FIG. 26 Gray's Anatomy plate|A: series of transverse sections through anembryo of the dog, anterior to posterior, I-V. Section I is the mostanterior. In V the neural plate is spread out nearly flat. Gray'sAnatomy by Henry Gray.

DEFINITIONS

As used herein, the term “inhibitor” in reference to inhibiting asignaling target or a signaling target pathway refers to a compound thatinterferes with (i.e. reduces or eliminates or suppresses) a resultingtarget molecule or target compound or target process, such as aparticular differentiation outcome, (for example, suppresses an activesignaling pathway promoting a default cell type differentiation, therebyinducing differentiation into a non-default cell type) when compared toan untreated cell or a cell treated with a compound that does notinhibit a treated cell or tissue.

As used herein, the term “Small Mothers Against Decapentaplegic” or“SMAD” refers to a signaling molecule.

As used herein, the term “neural cell” or “neuronal cell” refers to acell that in vivo would become part of the nervous system and in cultureis obtained by methods of the present inventions, for example, CNSprogenitor cells, patternable (i.e. a cell capable of undergoing furtherdifferentiation) neuronal populations of motorneurons and dopaminergicneurons, placodal precursor cells, high efficiency motor neuron cells,etc.

As used herein, the tem). “high efficiency motor neuron cell” refers toa neuronal cell capable of conducting an electric current.

As used herein, the term “fate” in reference to a cell, such as “cellfate determination” in general refers to a cell with a geneticallydetermined lineage whose progeny cells are capable of becoming a varietyof cell types or a few specific cell types depending upon in vivo or invitro culture conditions. In other words, a cell's predetermined fate isdetermined by it's environment to be destined for a particulardifferentiation pathway such that a cell becomes one cell type insteadof another cell type, for example, a stem cell's progeny cells whose“neural fate” is to become a nerve cell instead of a muscle cell or askin cell. Typically, a cell's “fate” is irreversible except underhighly specific conditions. In another example, a “CNS fate” refers to acell capable of becoming a cell associated with the central nervoussystem. Conversely, a cell fated to become a neural cell can be called a“neural progenitor cell.”

As used herein, the term “neural progenitor cell” refers to a cellcapable of forming a part of the nervous system, such as a nerve cell, aglial cell, etc.

As used herein, the term “neuronal subtype” refers to any cell of theneuronal system, such as a dopamine expression neuron, a peripherin+neuron, a motor neuron cell, etc.

As used herein, the term “cell of a neural lineage” refers to a cellthat differentiated along a neural precursor pathway.

As used herein, the term “placode” in reference to a cell refers to acell capable of becoming a cell associated with the sensory nervoussystem. In one embodiment, a placode cell is positive for Six1+,positive for p75 while negative for HNK1. In one embodiment, a placodecell obtained using methods of the present inventions is capable offorming a lens cell.

As used herein, the term “adenohypophyseal precursor” in reference to acell refers to a cell whose in vivo progeny cells would be or become apart of the pituitary gland. An adenohypophyseal precursor cell of thepresent inventions refers to a cell capable of expressing Lhx3 and CGA.

As used herein, the term “expressing” in relation to a gene or proteinrefers to making an mRNA or protein which can be observed using assayssuch as microarray assays, antibody staining assays, and the like.

As used herein, the term “inhibitor” in reference to inhibiting asignaling molecule or a signaling molecule's pathway, such as aninhibitor of SMAD signaling, refers to a compound that interferes with(i.e. reduces or eliminates or suppresses) the signaling function of themolecule or pathway. In one embodiment, an inhibitor of the presentinventions induces (changes) or alters differentiation from a default toa non-default cell type, for example, one of the methods of the presentinventions comprising two inhibitors of SMAD signaling produces anon-default neural progenitor cell.

As used herein, the term “Small Mothers Against Decapentaplegic” or“SMAD” refers to a signaling molecule.

As used herein, the term “cell differentiation” refers to a pathway bywhich a less specialized cell (i.e. stem cell) develops or matures topossess a more distinct form and function (i.e. neural plate).

As used herein, the term “differentiation” as used with respect to cellsin a differentiating cell system refers to the process by which cellsdifferentiate from one cell type (e.g., a multipotent, totipotent orpluripotent differentiable cell) to another cell type such as a targetdifferentiated cell.

As used herein, the term “cell differentiation” in reference to apathway refers to a process by which a less specialized cell (i.e. stemcell) develops or matures or differentiates to possess a more distinctform and/or function into a more specialized cell or differentiatedcell, (i.e. neural cell, neural plate cell, pituitary cell, adrenalcell, etc.).

As used herein, the term “neural stem cell” or “NSC” refers to a cellthat is capable of becoming neurons, astrocytes, oligodendrocytes, glialcells, etc., in vivo, and neuronal cell progeny and glial progeny inculture however their in vitro differentiation potential toward multipleregion-specific neuron types is low.

As used herein, the term “default” or “passive” in reference to a celldifferentiation pathway refers to a pathway where a less specializedcell becomes a certain differentiated cell type in culture, when nottreating with certain compounds i.e. normal cell cultures conditions. Inother words, a default cell results when a cell is not contacted by amolecule capable of changing the differentiated cell type (i.e. amorphogen). In contrast, “non-default” in reference to a cell refers toa differentiated cell type that results that is different from a defaultcell, i.e. a non-default cell is a differentiated cell type resultingfrom a non-default conditions, such as cell of the present inventions,including a dopamine positive nerve cell, a floor plate cell, posteriorFP tissue, etc. A default cell may also be a default cell after a cellhas contact with a morphogen to become a non-default cell without asubsequent morphogenic compound, such as a non-default floor plate cellthat subsequently becomes a default posterior FP cell of the non-defaultcell of the present inventions.

As used herein, the term “homodimer” in reference to a SMAD moleculerefers to at least two molecules of SMAD linked together, such as bydisulfide linkages.

As used herein, the term “Noggin” refers a secreted homodimericglycoprotein that binds to and inactivates members of the transforminggrowth factor-beta (TGF-β) superfamily of signaling proteins, such asbone morphogenetic protein-4 (BMP4). Noggin is typically a 65 kDaprotein expressed in human cells as a glycosylated, disulfide-linkeddimer. (Groppe, et al., (2002). Nature 420, 636-642; Xu, et al., (2005)Nat Methods 2, 185-190; Wang, et al., (2005) Biochem Biophys Res Commun330, 934-942). One example of a Noggin amino acid sequence is: Accession# U79163 single amino acid mouse Noggin (SEQ ID NO:50):

MERCPSLGVTLYALVVVLGLRAAPAGGQHYLHIRPAPSDNLPLVDFTLIEHPDPIFDPKEKDLNETLLRSLLGGHYDPGFMATSPPEDRPGGGGGPAGGAEDLAELFTDQLLRQRPSGAMPSEIKGLEFSEGLAQGKKQRLSKKLRRKLQMWLWSQTFCPVLYAWNDFTLGSRFWPRYVKVGSCFSKRSCSVPEGMVCKPSKSVHLTVLRWRCQRRGGQRCGWIPIQYFTPTISECKCSC.

As used herein, the term “SB431542” refers to a molecule with a numberCAS 301836-41-9, a molecular formula of C₂₂H₁₈N₄O₃, and a name of4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide,for example, see structure below:

As used herein, the term “Dorsomorphin” refers to a molecule with anumber CAS 866405-64-3, a molecular formula C₂₄H₂₅N₅O and a name of6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidinedihydrochloride, for example see structure below.

As used herein, the term “Lefty” refers to a novel member of thetransforming growth factor beta superfamily that inhibits TGF-beta,including but not limited to LEFTY1, LEFTY2, LEFTYA, etc., also known as“EBAF” or “endometrial bleeding associated factor” or “left-rightdetermination, factor A” transforming growth factor beta superfamily)).A Lefty protein is required for left-right asymmetry determination oforgan systems in mammals.

As used herein, the term “Activin” refers to a member of thetransforming growth factor-beta (TGF-β) superfamily, such as Activin A,Activin B, etc.

As used herein, the term “transforming growth factor beta” or “TGF-β”refers to a cytokine that regulates growth and differentiation ofdiverse types of cells.

As used herein, the term “nodal” refers to a member of the TGF-β familyof signaling molecules. Nodal signaling inhibits differentiation ofhuman embryonic stem cells along the neuroectodermal default pathway(Vallier, et al., Dev. Biol. 275, 403-421.

As used herein, the term “ALK” or “anaplastic lymphoma kinase” or“anaplastic lymphoma receptor tyrosine kinase” or “Ki-1” refers to amembrane associated tyrosine kinase receptor.

As used herein, the tem′ “ALK4” in reference to a type Iserine/threonine kinase receptor refers to an anaplastic lymphomareceptor tyrosine kinase 4 receptor that binds to activin to function asan activin receptor.

As used herein, the term “ALK5” in reference to a type Iserine/threonine kinase receptor refers to an anaplastic lymphomareceptor tyrosine kinase 5 receptor that binds to TGF-β1 to function asa TGF-β1 receptor.

As used herein, the tem′ “ALK7” in reference to a type Iserine/threonine kinase receptor refers to an anaplastic lymphomareceptor tyrosine kinase 7 receptor that binds to Nodal andNodal-related proteins to function as a Nodal and Nodal-related proteinreceptor.

As used herein, the term “paired box gene 6” or “PAX6” refers to amarker of a nondefault neuroprogenitor cell.

As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of cell differentiation, a kit mayrefer to a combination of materials for contacting stem cells, suchdelivery systems include systems that allow for the storage, transport,or delivery of reaction reagents (e.g., compounds, proteins, detectionagents (such as PAX6 antibodies), etc. in the appropriate containers(such as tubes, etc.) and/or supporting materials (e.g., buffers,written instructions for performing cell differentiation, etc.) from onelocation to another. For example, kits include one or more enclosures(e.g., boxes, or bags, and the like) containing the relevant reactionreagents (such as Noggin (or a Noggin substitute) and SB431542 (or aSB431542 replacement), etc.) and/or supporting materials.

As used herein, the term “inducing differentiation” in reference to acell refers to changing the default cell type (genotype and/orphenotype) to a non-default cell type (genotype and/or phenotype). Thus“inducing differentiation in a stem cell” refers to inducing the cell todivide into progeny cells with characteristics that are different fromthe stem cell, such as genotype (i.e. change in gene expression asdetermined by genetic analysis such as a microarray) and/or phenotype(i.e. change in expression of a protein, such as PAX6 or a set ofproteins, such as HMB45 positive (+) while negative (−) for SOX10.

As used herein, the term “contacting” cells with a compound of thepresent inventions refers to placing the compound in a location thatwill allow it to touch the cell in order to produce “contacted” cells.The contacting may be accomplished using any suitable method. Forexample, in one embodiment, contacting is by adding the compound to atube of cells. Contacting may also be accomplished by adding thecompound to a culture of the cells.

As used herein, the tem “stem cell” refers to a cell that is totipotentor pluripotent or multipotent and are capable of differentiating intoone or more different cell types, such as embryonic stems cells, stemcells isolated from organs, for example, skin stem cells.

As used herein, the term “embryonic stem cell” refers to a cell of astem cell line, such as WA-09, or a cell isolated from an embryo orplacenta or umbilical cord.

As used herein, the term “adult stem cell” refers to a stem cell derivedfrom an organism after birth.

As used herein, the term “neural stem cell” or “NSC” refers to a cellthat is capable of becoming neurons, astrocytes, oligodendrocytes, andglial cells in vivo, and neuronal cell progeny and glial progeny inculture however their in vitro differentiation potential toward multipleregion-specific neuron types is low.

As used herein, the term “mesodermal cell line” refers to a cell linedisplaying characteristics associated with mesodemial cells.

As used herein, the term “endodermal cell line” refers to a cell linedisplaying characteristics normally associated with endodermal cells.

As used herein, the term “neural cell line” refers to a cell linedisplaying characteristics normally associated with a neural cell.Examples of such characteristics include, but are not limited to,expression of FOXA2, SHH, Netrin-1, F-Spondin, and the like.

As used herein, the term “totipotent” refers to an ability of a cell todifferentiate into any type of cell in a differentiated organism, aswell as a cell of extra embryonic materials, such as placenta, etc.

As used herein, the term “pluripotent” refers to a cell line capable ofdifferentiating into any differentiated cell type.

As used herein, the term “multipotent” refers to a cell line capable ofdifferentiating into at least two differentiated cell types.

As used herein, the term “differentiation” as used with respect to cellsin a differentiating cell system refers to the process by which cellsdifferentiate from one cell type (e.g., a multipotent, totipotent orpluripotent differentiable cell) to another cell type such as a targetdifferentiated cell.

As used herein, the term “cell culture” refers to any in vitro cultureof cells. Included within this term are continuous cell lines (e.g.,with an immortal phenotype), primary cell cultures, finite cell lines(e.g., non-transformed cells), and any other cell population maintainedin vitro, including oocytes and embryos.

As used herein, the term “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments can consist of, but are not limitedto, test tubes and cell cultures. The term “in vivo” refers to thenatural environment (e.g., an animal or a cell) and to processes orreaction that occur within a natural environment.

As used herein, the term “neural plate” or “medullary plate” refers to athickened band of ectoderm (an unpaired ventral longitudinal zone of theneural tube) in the midbody region of the developing embryo, whichdevelops (differentiates) into the neural tube and neural crest; see,FIG. 12. During embryonic development neural plate cells undergo aseries of developmental stages and subsequently develop into cellsforming a brain, spinal cord, and other tissues of the central nervoussystem.

As used herein, the term “floor plate” or “FP” or “ventral plate” or“basal plate” or “neural floor plate” refers to an area of cells thatdevelops at the midline of the neural plate and is located at theventral midline of the neural tube, see, FIG. 12.

As used herein, the term “neural floor plate cell” or “FP cell” or“floor plate cell” in reference to a cell refers to a cell group alsocalled “specialized neuroepithelial cells” found in a developing embryoin the neural floor plate. FP cell in vitro are cells expressing certaincell markers also found in FP cells in vivo that are not found in othercells.

As used herein, the term “roof plate” or “alar plate” or “dorsal roofplate” refers to a cell group located in at the dorsal region of theforming and formed neural tube areas the unpaired dorsal longitudinalzone of the neural tube.

As used herein, the term “neural tube” refers to a hollow cylindricalstructure of neuroepithelial cells formed from the neuroectoderm cellsof an early embryo by the closure of the neural groove such thatneuroectoderm cells can differentiate into brain cells and spinal cordcells.

As used herein, the term “presumptive” or “progenitor” in reference to acell or an area of cells refers to the type of cell or area of cellsthat would develop (differentiate into) under the appropriateconditions, i.e. when contacted with a proper growth factor, compound,extracellular signal, intracellular signal, etc. For example,“progenitor neuron” refers to a cell that has the capability to developinto a neuron.

As used herein, the term “dopamine neuron” or “dopaminergic neuron” ingeneral refers to a cell capable of expressing dopamine. “Midbraindopamine neurons” or “mDA” refer to presumptive dopamine expressingcells in forebrain structures and dopamine expressing cells in forebrainstructures.

As used herein, the term “default” in reference to a celldifferentiation pathway refers to a pathway where a less specializedcell becomes a certain differentiated cell type when not contacted by amolecule which changes the differentiated cell type.

As used herein, the term “cell differentiation” refers to a pathway bywhich a less specialized cell (i.e. stem cell) develops or matures topossess a more distinct form and function (i.e. neural plate).

As used herein, the term “neurite outgrowth refers to observation ofelongated, membrane-enclosed protrusions of cytoplasm from cells.

As used herein, the term “attached cell” refers to a cell growing invitro wherein the cell contacts the bottom or side of the culture dish,an attached cell may contact the dish via extracellular matrix moleculesand the like. As opposed to a cell in a suspension culture.

As used herein, the term “marker” or “cell marker” refers to gene orprotein that identifies a particular cell or cell type. A marker for acell may not be limited to one marker, markers may refer to a “pattern”of markers such that a designated group of markers may identity a cellor cell type from another cell or cell type. For example, FP cells ofthe present inventions express one or more markers that distinguish a FPcell, i.e. FOXA2 positive and BF1 negative, from a nonFP cell, i.e.FOXA2 negative and BF1 positive.

As used herein, the term “test compound” refers to any chemical entity,pharmaceutical, drug, and the like that were used to provide cells ofthe present inventions.

As used herein, the term “rosette-stage neural cell” or “R-NSC” refersto a neural stem cell type in vitro with broad differentiation potentialcapable of forming central nervous system (CNS) and peripheral nervoussystem (PNS) cells (fates) and capable of in vivo engraftment. In otherwords, a rosette-stage neural cell is capable of forming a rosettestructure and rosette-stage neural cell populations have characteristicsof neuronal differentiation.

As used herein, the term “rosette structure” or “rosette” in referenceto a cell refers to a halo or spoke-wheel arrangement of cells.

As used herein, the term “increasing” in reference to a characteristicrefers to a larger amount of a characteristic when compared to saidcharacteristic in a control, such as when comparing an amount of amarker in human embryonic stems cells cultured with and without a testcompound.

As used herein, the term “decreasing” in reference to a characteristicrefers to a smaller amount of a characteristic when compared to saidcharacteristic in a control, such as when comparing an amount of amarker in human embryonic stems cells cultured with and without a testcompound.

As used herein, the term “reducing protein function” or “loss offunction” refers to interfering with or blocking a function in order tolower that function, for example, lowering the function of DKK-1 byblocking antibodies.

As used herein, the term “neuron inducing compound” refers to asubstance for causing differentiation along a cellular pathway leadingto neuronal cell.

As used herein, the term “agent for blocking phosphorylation of areceptor” refers to a substance for reducing receptor function, i.e. byreducing phosphorylation.

As used herein, the term “response,” when used in reference to an assay,refers to the generation of a detectable signal (e.g., accumulation ofreporter protein, increase in ion concentration, accumulation of adetectable chemical product).

The term “sample” is used in its broadest sense. In one sense it canrefer to a cell or tissue. In another sense, it is meant to include aspecimen or culture obtained from any source and encompass fluids,solids and tissues. Environmental samples include environmental materialsuch as surface matter, soil, water, and industrial samples. Theseexamples are not to be construed as limiting the sample types applicableto the present invention.

The terms “purified,” “to purify,” “purification,” “isolated,” “toisolate,” “isolation,” and grammatical equivalents thereof as usedherein, refer to the reduction in the amount of at least one contaminantfrom a sample. For example, a cell type is purified by at least a 10%,preferably by at least 30%, more preferably by at least 50%, yet morepreferably by at least 75%, and most preferably by at least 90%,reduction in the amount of undesirable cell types, such as isolateddifferentiated FP cells from nonFP cells, such as cells present in amixed cell culture. Thus purification of a cell type results in an“enrichment,” i.e., an increase in the amount, of the nucleotidesequence in the sample.

The term “naturally occurring” as used herein when applied to an object(such as cell, tissue, etc.) and/or chemical (such as a protein, aminoacid sequence, nucleic acid sequence, codon, etc.) means that the objectand/or compound are/were found in nature. For example, a naturallyoccurring cell refers to a cell that is present in an organism that canbe isolated from a source in nature, such as an embryonic cell, whereinthe cell has not been intentionally modified by man in the laboratory.

As used herein the term, “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments exemplified, but are not limited to,test tubes and cell cultures.

As used herein the term, “in vivo” refers to the natural environment(e.g., an animal or a cell) and to processes or reactions that occurwithin a natural environment, such as embryonic development, celldifferentiation, neural tube formation, etc.

As used herein, the term “proliferation” refers to an increase in cellnumber.

As used herein, the term “ligand” refers to a molecule that binds to asecond molecule. A particular molecule may be referred to as either, orboth, a ligand and second molecule. Examples of second molecules includea receptor of the ligand, and an antibody that binds to the ligand.

The term “derived from” or “established from” or “differentiated from”when made in reference to any cell disclosed herein refers to a cellthat was obtained from (e.g., isolated, purified, etc.) a parent cell ina cell line, tissue (such as a dissociated embryo, or fluids using anymanipulation, such as, without limitation, single cell isolation,cultured in vivo, treatment and/or mutagenesis using for exampleproteins, chemicals, radiation, infection with virus, transfection withDNA sequences, such as with a morphagen, etc., selection (such as byserial culture) of any cell that is contained in cultured parent cells.A derived cell can be selected from a mixed population by virtue ofresponse to a growth factor, cytokine, selected progression of cytokinetreatments, adhesiveness, lack of adhesiveness, sorting procedure, andthe like.

As used herein, the term “biologically active,” refers to a molecule(e.g. peptide, nucleic acid sequence, carbohydrate molecule, organic orinorganic molecule, and the like) having structured, regulatory, and/orbiochemical functions.

As used herein, the term “primary cell” is a cell that is directlyobtained from a tissue (e.g. blood) or organ of an animal in the absenceof culture. Typically, though not necessarily, a primary cell is capableof undergoing ten or fewer passages in vitro before senescence and/orcessation of proliferation. In contrast, a “cultured cell” is a cellthat has been maintained and/or propagated in vitro for ten or morepassages.

As used herein, the term “cultured cells” refer to cells that arecapable of a greater number of passages in vitro before cessation ofproliferation and/or senescence when compared to primary cells from thesame source. Cultured cells include “cell lines” and “primary culturedcells.”

As used herein, the term “cell culture” refers to any in vitro cultureof cells. Included within this term are continuous cell lines (e.g. withan immortal phenotype), primary cell cultures, finite cell lines (e.g.,non-transformed cells), and any other cell population maintained invitro, including embryos and embryonic cells.

As used herein, the term “cell line,” refers to cells that are culturedin vitro, including primary cell lines, finite cell lines, continuouscell lines, and transformed cell lines, but does not require, that thecells be capable of an infinite number of passages in culture. Celllines may be generated spontaneously or by transformation.

As used herein, the turns “primary cell culture,” and “primary culture,”refer to cell cultures that have been directly obtained from cells invivo, such as from animal tissue. These cultures may be derived fromadults as well as fetal tissue.

As used herein, the terms “monolayer,” “monolayer culture,” and“monolayer cell culture,” refers to a cell that has adhered to asubstrate and grow as a layer that is one cell in thickness, in otherwords, an “attached cell.” Monolayers may be grown in any format,including but not limited to flasks, tubes, coverslips (e.g., shellvials), roller bottles, et cetera.

As used herein, the terms “feeder cell layer” or “feeder cellpopulation” refers to a monolayer of cells used to provide attachmentmolecules and/or growth factors for an adjacent cell, for example, usedin co-culture to maintain pluripotent stem cells. As used herein, theterms “suspension” and “suspension culture” refer to cells that surviveand proliferate without being attached to a substrate. Suspensioncultures are typically produced using hematopoietic cells, transformedcell lines, and cells from malignant tumors.

As used herein, the terms “culture media,” and “cell culture media,”refer to media that are suitable to support the growth of cells in vitro(i.e., cell cultures, cell lines, etc.). It is not intended that theterm be limited to any particular culture medium. For example, it isintended that the definition encompass outgrowth as well as maintenancemedia. Indeed, it is intended that the think encompass any culturemedium suitable for the growth of the cell cultures and cells ofinterest.

The term, “cell biology” or “cellular biology” refers to the study of alive cell, such as anatomy and function of a cell, for example, a cell'sphysiological properties, structure, organelles, interactions with theirenvironment, their life cycle, division and death.

As used herein, the term “cell” refers to a single cell as well as to apopulation of (i.e., more than one) cells. The population may be a purepopulation comprising one cell type, such as a population of neuronalcells or a population of undifferentiated embryonic cells.Alternatively, the population may comprise more than one cell type, forexample a mixed cell population. It is not meant to limit the number ofcells in a population, for example, a mixed population of cells maycomprise at least one differentiated cell. In one embodiment a mixedpopulation may comprise at least one differentiated. In the presentinventions, there is no limit on the number of cell types that a cellpopulation may comprise.

As used herein, the term “positive cell” in relation to a stain refersto a cell that expresses a marker and thus “stains” for that marker in adetectable quantitative and/or qualitative amount above a control orcomparative cell. A positive cell may also refer to a cell that stainsfor a molecule such as FOXA2, et cetera.

As used herein, the term “negative cell,” refers to a cell absentdetectable signal for a marker, such as a cell failing to stainfollowing contacting with a FOXA2 antibody detection method, et cetera.

As used herein, the tem “caudalization” refers to initiation ofposterior pathways of neural development in the dorsalized ectodermduring embronic development, for example, dorsalized ectoderm developsvarious levels of posterior neural tissues, depending on the extent ofcaudalization.

As used herein, the term “caudalizing agent” or “caudalizing factor”refers to a compound that induces caudalization, such as Wnt-1 andRetinoic Acid (RA)

As used herein, the terms “reporter gene” or “reporter construct” referto genetic constructs comprising a nucleic acid encoding a protein thatis easily detectable or easily assayable, such as a colored protein,fluorescent protein or enzyme such as beta-galactosidase (lacZ gene).

As used herein, the term “gene targeting” refers the integration ofexogenous DNA into the genome of a cell at sites where its expressioncan be suitably controlled. This integration occurs as a result ofhomologous recombination.

A “knock-in” approach as used herein refers to the procedure ofinserting a desired nucleic acid sequence, such as a sequence encoding areporter gene, into a specific locus in a host cell via homologousrecombination.

As used herein, the term “genome” refers to the genetic material (e.g.,chromosomes) of an organism.

The term “nucleotide sequence of interest” refers to any nucleotidesequence (e.g., RNA or DNA), the manipulation of which may be deemeddesirable for any reason (e.g., treat disease, confer improvedqualities, expression of a protein of interest in a host cell,expression of a ribozyme, etc.), by one of ordinary skill in the art.Such nucleotide sequences include, but are not limited to, codingsequences of structural genes (e.g., reporter genes, selection markergenes, oncogenes, drug resistance genes, growth factors, etc.), andnon-coding regulatory sequences which do not encode an mRNA or proteinproduct (e.g., promoter sequence, polyadenylation sequence, terminationsequence, enhancer sequence, etc.).

As used herein, the term “protein of interest” refers to a proteinencoded by a nucleic acid of interest.

As used herein, the term “exogenous gene” refers to a gene that is notnaturally present in a host organism or cell, or is artificiallyintroduced into a host organism or cell.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises coding sequences necessary for the production of apolypeptide or precursor (e.g., proinsulin). The polypeptide can beencoded by a full length coding sequence or by any portion of the codingsequence so long as the desired activity or functional properties (e.g.,enzymatic activity, ligand binding, signal transduction, etc.) of thefull-length or fragment are retained. The terms also encompasses thecoding region of a structural gene and includes sequences located,adjacent to the coding region on both the 5′ and 3′ ends for a distanceof about 1 kb or more on either end such that the gene corresponds tothe length of the full-length mRNA. The sequences that are located 5′ ofthe coding region and which are present on the mRNA are referred to as5′ untranslated sequences. The sequences that are located 3′ ordownstream of the coding region and which are present on the mRNA arereferred to as 3′ untranslated sequences. The term “gene” encompassesboth cDNA and genomic forms of a gene. A genomic form or clone of a genecontains the coding region interrupted with non-coding sequences termed“introns” or “intervening regions” or “intervening sequences.” Intronsare segments of a gene that are transcribed into nuclear RNA (hnRNA);introns may contain regulatory elements such as enhancers. Introns areremoved or “spliced out” from the nuclear or primary transcript; intronstherefore are absent in the messenger RNA (mRNA) transcript. The mRNAfunctions during translation to specify the sequence or order of aminoacids in a nascent polypeptide.

As used herein, the term “gene expression” refers to the process ofconverting genetic information encoded in a gene into RNA (e.g., mRNA,rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e., via theenzymatic action of an RNA polymerase), and for protein encoding genes,into protein through “translation” of mRNA. Gene expression can beregulated at many stages in the process. “Up-regulation” or “activation”refers to regulation that increases the production of gene expressionproducts (i.e., RNA or protein), while “down-regulation” or “repression”refers to regulation that decrease production. Molecules (e.g.,transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” “DNA encoding,” “RNA sequence encoding,” and “RNAencoding” refer to the order or sequence of deoxyribonucleotides orribonucleotides along a strand of deoxyribonucleic acid or ribonucleicacid. The order of these deoxyribonucleotides or ribonucleotidesdetermines the order of amino acids along the polypeptide (protein)chain. The DNA or RNA sequence thus codes for the amino acid sequence.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to the field of cell biology ofstem cells, more specifically the directed differentiation ofpluripotent or multipotent stem cells, including human embryonic stemcells (hESC), somatic stem cells, and induced human pluripotent stemcells (hiPSC) using novel culture conditions. Specifically, methods areprovided for obtaining neural plate floor tissue and floor plate cellsincluding induction of neural plate floor development in hESCs forobtaining dopamine (DA) nerve cells. Further, neural plate floor tissueobtained using methods of the present inventions are contemplated foruse in co-cultures with other tissues as inducers for shiftingdifferentiation pathways, i.e. patterning.

Transforming growth factor (TGF-beta) and their family members,including bone morphogenetic proteins (BMPs), Nodal, and activins, areinvolved in the development and maintenance of various organs, in whichstem cells play important roles. The ectoderm germ layer of the embryogives rise to the neuroectoderm (the central and peripheral nervoussystem, neural crest cells, and derivatives). Several lines of evidencedemonstrate a crucial role for Smad signaling during neural induction.Generally, Smad proteins are downstream of TGF-beta superfamily ligands,and when their specific receptors are activated, they stimulate thephosphorylation of the receptor-regulated Smads (R-Smads: Smad1, Smad2,Smad3, Smad5 and Smad 8, with Smads 2 and 3 specifically activated byactivin/nodal and TGF-beta type I receptors and Smads 1, 5, and 8activated by BMP type I receptors). Two distinct pathways converse onSmad 4.

Elegant studies in frog identified BMP inhibitors including chordin(Sasai, et al., Cell 79(5):779 (1994)), follistatin (Hemmati-Brivanlou,et al., Cell 77(2):283 (1994)), and noggin (Smith, et al., Cell70(5):829 (1992)) as the critical neural inducing factors in theSpearman organizer. Mammalian noggin (Valenzuela, et al., J Neurosci15(9):6077 (1995)) has comparable neural inducing properties, andtreatment with recombinant Noggin has been used in several hESC neuralinduction protocols (Lee, et al., Stem Cells 25(8):1931 (2007);Elkabetz, et al., Genes Dev 22(2):152 (2008)). More recently, the drugSB431542 was shown to enhance neural induction in an embryoid body (EB)based hESC neural induction protocol (Smith, et al., Dev Biol 313(1):107(2008)). SB431542 inhibits the Lefty/Activin/TGFβ pathways by blockingphosphorylation of ALK4, ALK5, ALK7 receptors. While Noggin or SB431542treatment improve the efficiency of neural induction, neither treatmentalone is sufficient to neurally convert hESCs under defined or adherentconditions.

Efforts to culture stem cells under conditions that are robust andhighly repeatable, and minimize opportunities of cross contamination.The efforts towards establishing well-defined media and methods willallow for repeatability and accuracy required for use as a therapeuticagent, and there has been a move to establish media and cultureconditions that are free of non-human additives and undefined factors.

Modifications of the cell culture system have been the focus of a numberof recent patents. U.S. Pat. No. 7,005,252, herein incorporated byreference, discusses the growth of primate embryonic stem cells in thepresent of Fibroblast Growth Factor and a feeder cell layer, but in theabsence of any animal serum. U.S. Pat. No. 7,297,539, hereinincorporated by reference, discusses the growth of pluripotent stemcells utilizing a system containing an extracellular matrix in aFibroblast Growth Factor containing medium, but without a feeder layer.U.S. Pat. No. 7,211,434, herein incorporated by reference, describes amethod for culturing mammalian embryonic stem cells in a serum for freeand feeder-layer free media containing leukemia inhibitory factor,another cytokine used to maintain pluripotency. Identification ofspecific and defined compounds as additives to media to control the fateof embryonic stem cells are the focus of a number of used patents,including U.S. Pat. Nos. 7,332,336, 7,294,510 and 7,252,995, each ofwhich are herein incorporated by reference.

Human stem cells offer great promise for cell-replacement therapies, andrecent advances in somatic cell reprogramming to induced pluripotentstem cells (hiPSCs) has opened the door to generating patient-specificcells for regenerative medicine and disease modeling (Takahasi et al,2007; Kim, et al., Cell, 136(3):411-419 (2009)). However to realize thefull potential of these approaches, improved differentiation protocolsare required that eliminate the use of undefined factors such asneural-inducing stoma (PAX6 or MS5 cells (Kawasaki et al., 2000; Lee etal 2007)), the heterogeneous nature of EB differentiation of the pooryield of protocols based on selective survival of neural progeny.Understanding and selectively triggering the signaling pathwaysnecessary and sufficient for neural indication in hESCs is a criticalgoal in this effort.

Neural stem cell progenitors and neural subtypes as derived from stemcells have been the focus of numerous scientific publications and patentapplications, but threes disclosures lack the most desirable conditionsfor controlling stem cell fate including the ability to start with alarge number of cells, achieve highly homogenous desired cell fates, anduse a feeder-free protocol and under adherent conditions. Shang et al.,and Reubinoff, et al., Nature Biotechnology 19, 1134-1140 (2001) allowfor passive development of neural cell types, but cannot control theneural differentiation.

United States Patent Application Publication No. 20090035285, hereinincorporated by reference, teaches methods of producing neural cells,relying on EB and rosette formation. U.S. Pat. No. 6,833,269, hereinincorporated by reference, provides differentiation of cells rely on theuse of feeder cells and EB formation. U.S. Pat. No. 7,011,828, hereinincorporated by reference, and Application Publication No. 2005026747,herein incorporated by reference, teaches and 20060078543, hereinincorporated by reference, teach the proliferation of an enrichpopulation of hESCs, which are differentiated to neural progenitorcells, neurons, or glial cells. U.S. Pat. No. 6,887,706, hereinincorporated by reference, teaches a method of differentiating heESCsintor neural precursor cells using FGF2, whereby in vitrodifferentiation was induced by withdrawal of FGF2 and plating onornithine and laminin substrate. U.S. Pat. No. 7,368,115 teachesdifferentiation of neural stem cells or neural stem cell progeny withpituitary adenylate cyclase-activating polypeptide.

In a review by Erceg et al., Stem Cells. January; 27(1): 78-87 (2009),herein incorporated by reference, the author noted that the mostimportant concern of the recently published protocols of stem celldifferentiation towards neural lineages is (i) the risk of non-neuralcell contamination; (ii) that the use of stem cell lines, Matrigel orconditioned media, including procedures relying on EB formation bearsthe risk of pathogen cross-transfer. None of the foregoing patents orpatent applications teaches the derivatization of homogenous populationof neural cell lineage from stem cells under the conditions present inthis invention.

The present invention further relates to methods of obtainingpopulations of neural progenitor cells derived from human embryonic stemcells (hESCs), in particular for obtaining neural plate floor tissue.Specifically, methods of the present inventions induce neural platefloor development in hESCs for obtaining dopamine (DA) nerve cells.Further, neural plate floor tissue obtained using methods of the presentinventions are contemplated for use in co-cultures with other tissues asinducers for shifting differentiation pathways, i.e. patterning.

Current neural induction protocols in human ES cells (hESs) relyembryoid body formation, stromal feeder co-culture, or selectivesurvival conditions; each strategy displaying significant drawbacks suchas poorly defined culture conditions, protracted differentiation and lowyield.

Synergistic action of two inhibitors of SMAD signaling was discovered bythe inventors and reported herein. Noggin and SB431542 were discoveredto be sufficient for inducing rapid and completed neural conversion ofhESCs under adherent culture conditions (dual SMAD inhibition protocol).Temporal fate analysis reveals a transient FGF5+ epiblast-like stagefollowed by PAX6+ neural cells competent of rosette formation. Initialcell density determines the ratio of CNS versus neural crest progeny.Directed differentiation of huPSCs into midbrain dopamine and spinalmotor neurons confirm robustness and general applicability of the novelinduction protocol. Noggin/SB4315242 based neural induction shouldgreatly facilitate the use of hESC and hiPSCs in regenerative medicineand disease modeling and obviate the need for stromal feeder or EB-basedprotocols. Further, this method were adapted to culture systems whichmay enhance the ease, yield efficiency, speed at which neural cells arederived. This should not be considered limiting and culture withadditional molecules or growth factors, or incorporating other methodsin the art were considered.

Several lines of evidence demonstrate a crucial role for SMAD signalingduring neural induction. While Noggin or SB431542 treatment improve theefficiency of neural induction, neither treatment alone is sufficient toneurally convert hESCs under defined or adherent conditions. Here Theinventors set out to test whether combined blockade of SMAD signalingusing Noggin and SB431542 is sufficient to achieve full neuralconversion and to obviate the need for EB- or stromal-feeder basedprotocols.

The inventors contemplated that establishing an even cell distributionis critical for inducing homogenous neural differentiation of hESCs. Tothis end, undifferentiated hESC were dissociated into single cells andre-plated onto Matrigel coated dishes in conditioned medium supplementwith ROCD inhibitor, Y-27632 (Wananabe et at, 2007). After 72 hourscells were switched from hESC conditions to knock-out serum replacementmedium (KSR) containing either Noggin, SB4315242, or both factors andallowed to differentiate for a total of 11 days (FIG. 1.a.). Thereduction in nuclear localization of the oblige co-Smad, Smad 4, wasobserved after 24 hours when both Noggin and SB431542 were present (FIG.2). Neural induction was monitored by expression of PAX6, an earlymarker of neuroectodermal differentiation (Callaerts, et al., Annu RevNeurosci 20:483 (1997).

Combined treatment with Noggin and SB431542 dramatically increased theefficiency of neural induction to greater than 80% of total cells,compared with less than 10% PAX6⁺ cells when Noggin or SB431542 wereused alone (FIG. 1b ). The synergistic action is unexpected, and thereare several potential mechanisms that could contribute to thesynergistic action of noggin and SB431542. These include destabilizingthe activin- and Nanog-mediated pluripotency network (Xu, et al., CellStem Cell 3(2):196 (2008)), suppression of BMP induced differentiationtowards trophoblast lineage (Xu, et al., Nat Biotechnol 20(12):1261(2002)), suppression of mes/endodermal fates by inhibiting endogenousactivin and BMP signals (D'Amou, et al., Nat Biotechnol 23(12):1534(2005)) and promoting neuralization of primitive ectoderm by BMPinhibition (Laflamme, et al., Nat Biotechnol 25(9):1015 (2007)).

Temporal analysis of gene expression revealed that treatment withSB431542 induced a rapid loss of Nanog expression (FIG. 4) and adramatic increase in the expression of CDX2 (FIG. 1c ). These datasuggested SB431542 mediated loss of pluripotency is associated withdifferentiation towards trophoblast lineage. Suppression of CDX2 in thepresence of Noggin or Noggin/SB431542 demonstrates that one key role ofNoggin is the repression of endogenous BMP signals that drivetrophoblast fates upon differentiation. The pronounced induction of SOX1in Noggin/SB431542 treated cultures confirmed a strong bias towardsneuroectodermal lineage in the dual SMAD inhibition protocol. There isalso evidence for suppression of alternative embryonic germ layers suchas Noggin-mediated suppression of SOX17 (endodermal lineage) andSB431542 mediated suppression of Brachyury (mesodermal lineage) (FIG. 1c). Taken together, these results indicate that SB431542 and Noggin worksynergistically at multiple stages of differentiation to achieveefficient neural conversion of hESCs.

Next lineage progression of hESC progeny after the addition of the twoinhibitors was characterized. Immunocytochemical analysis showed loss ofOCT4 expression by day 5 and strong expression of PAX6 by day 7 (FIG. 1d). These data pointed to the presence of an intermediate cell type atday 5 of differentiation that was negative for both OCT4 and PAX6. Geneexpression analysis revealed peak expression of the epiblast marker FGF5at day 5 of differentiation concomitant with high expression of Otx2,another epiblast marker whose expression is maintained during neuralfate commitment (FIG. 1e ). Interestingly, the earliest neural markerexpressed in our culture system was SOX1 (FIG. 1f ), preceding inductionof other neuroepithelial markers such as ZIC1 or PAX6, and precedingexpression of anterior CNS (FOXG1) and neural crest (p75) markers. Earlyinduction of SOX1 is distinct from previous studies that had suggestedPAX6 expression preceding SOX1 induction (Munoz-Sanjuan, et al., Nat RevNeurosci 3(4):271 (2002)). One interesting possibility to explain thisdiscrepancy could be direct modulation of SOX1 transcription by SMADsignaling in our culture system. Recently methods for were described forestablishing stable mouse (Munoz-Sanjuan, et al., Nat Rev Neurosci3(4):271 (2002)) and hESC (Placantonakis, et al., Stem Cells27(3):521-532 (2009)) transgenic reporter lines carrying bacterialartificial chromosomes (BACs) engineered to express GFP under control ofcell type specific promoters. Here The inventors used the HES5::eGFP BACtransgenic hESC reporter line, marking neural stem and precursor cellprogeny (Tesar, Nature 448:196-199 (2007); (Placantonakis, et al., StemCells 27(3):521-532 (2009)), to measure the efficiency of neuralinduction. The dual SMAD inhibition protocol was compared to thestandard MS5 protocol in the presence of Noggin (Perrier, et al., ProcNatl Acad Sci USA 101(34):12543 (2004)). To this end HES5::eGFP cellswere plated in media supplemented with Noggin either in the presence ofMS5 feeder cells or SB431542 and allowed to differentiate for 13 days, astage when the GFP⁺ cells were readily observed under both conditions(FIG. 4). GFP expression was quantified by flow cytometry. Non-modifiedH9 cells were used as negative controls. MS5 cells were excluded fromthe analysis based on negative selection for the cell surface moleculeCD105 (FIG. 5). Dual SMAD inhibition yielded 82% GFP⁺ cells at day 13, amore than 3 fold increase compared with the MS5/Noggin protocol (FIG. 1f).

In contrast to the MS5 protocol which requires plating of hESC coloniesat low density (Li, et al., Nat Biotechnol 23(2):215 (2005)), theNoggin/SB431542 condition allowed for high plating densities. Therefore,in addition to higher percentages, the dual SMAD inhibition protocolalso resulted in larger absolute numbers of Hes5::eGFP⁺ cells per eachculture plate.

(Isolation of rosette neural stem cells was reported (Elkabetz, et al.,Genes Dev, 22, 152-165 (2008)) (R-NSCs) in addition to development ofneural crest stem cells (Lee, et al., Stem Cells 25 (8), 1931 (2007))(NCSCs) from hESCs. The inventors next sought to determine the lineagerelationship of the early PAX6⁺ neuroectodermal cells observed in thedual SMAD inhibition protocol to the R-NSCs and NCSCs populationsdescribed previously. Immunocytochemical analysis showed that, similarto R-NSCs, PAX6⁺ neuroectodermal cells express general NSC markers suchas Nestin and R-NSC markers including PLZF (FIGS. 2 a and b; day 11 ofdifferentiation). However, cytoarchitecture and ZO1 expression indicatedthat neuroepithelial cells under these conditions were non-polarizedexhibiting an ESC-like cytoarchitecture. These non-polarized areas wereinterspersed with R-NSC like areas composed of polarized columnarepithelial cells (FIG. 7c ). The developmental hierarchy of these twocell populations was further explored upon subsequent passage. Underthese conditions early neuroepithelial cells spontaneously convertedinto rosette structures with apical ZO1 expression and evidence ofinterkinetic nuclear migration (FIG. 2d ). These data suggested that theNoggin/SB431542 protocol yields an early PAX6⁺ neuroepithelialpopulation capable of rosette formation. The early PAX6⁺ cells maytherefore represent the most primitive hESC derived neural precursorstage isolated to date. R-NSCs have been shown to acquire anterior CNSmarker by default (Elkabetz, et al., Genes Dev. 22:152-165 (2008)).PAX6⁺ neuroepithelial cells generated via the dual SMAD inhibitionprotocol exhibited an anterior CNS character as evidenced by expressionof Otx2 and FoxG1B (FIG. 2 e, f) similar to R-NSCs (Elkabetz, et al.,Genes Dev. 22:152-165 (2008)). Interestingly, PAX6 negative cells underthese conditions co-expressed markers of neural crest including AP2,HNK1, PAX7, and p75 (NGFR) (FIG. 2g-j ). Manipulations of the initialhESC plating density skewed the ratio of PAX6⁺ CNS versus PAX6⁻ neuralcrest-like cells. High plating densities resulted in near exclusivedifferentiation towards PAX6⁺ cells while low densities promoted neuralcrest-like differentiation (FIG. 6). The presence of large numbers ofneural crest-like cells prior to rosette formation suggested that dualSMAD inhibition yields an early neural crest population distinct fromR-NSC derived NCSCs (Lee, et al., Stem Cells 25(8):1931 (2007)).Supporting the notion of an early neural crest population with distinctlineage potential cells could be readily enriched for pigmented cellsco-expressing the melanosome marker, HMB45 (FIGS. 2 k and l, seeExamples for details). In contrast, R-NSC derived NCSCs typically do notyield pigmented cells under comparable conditions (Lee, et al., StemCells 25(8):1931 (2007)). However, some HMB45⁺ cells did not coexpressthe neural crest marker SOX10 suggesting the presence of other pigmentedcell populations including PAX6⁺ retinal pigment epithelial cells.

Anterior-posterior (AP) and dorso-ventral (DV) identity and neuronalsubtype potential is dependent on early exposure to morphogenic factorssuch as retinoic acid, FGF8, and SHH. The inventors next explored thepatterning potential of cells generated via the dual SMAD inhibitionprotocol. The inventors postulated that day 5 of differentiation maypresent an appropriate developmental window for neural patterning sinceOct4 expression is silenced between day 3 and 5 and the neural markerPAX6 is activated in the majority of cells between day 5 and 7 (FIG. 1d,e ). Derivation of cells expressing markers of dopamine neurons wasobserved following exposure to SHH and FGF8 (Tomishima, et al., StemCells 25 (1), 39 (2007)) starting at day 5 and day 9 of differentiationrespectively (FIG. 2m ). One week after SHH exposure, both FGF8 and SHHwere withdrawn and further differentiated in medium containing BDNF,ascorbic acid, GDNF, TGF-β3, and cyclic-AMP (BAGTC (Tomishima, et al.,Stem Cells 25 (1), 39 (2007)), see FIG. 2m ). At day 19 ofdifferentiation neurons a large proportion of Tuj1⁺ neurons co-expressedtyrosine hydroxylase (TH) (FIG. 2n, o ), the rate-limiting enzyme in thesynthesis of dopamine. TH⁺ neurons emerged under these conditionsspontaneously even in the absence of cell passaging. However, derivationof more mature TH⁺ cells with long neural processes was promotedfollowing mechanical isolation and en bloc passage at day 12 ofdifferentiation.

Nuclear expression of the motor neuron markers ISL1 and HB9 was observedtwo weeks upon exposure to BDNF, ascorbic acid, SHH, and retinoic acid(BASR; day 19 of differentiation) confirming the derivation of somatictype motor neurons (FIG. 2q,r ). Motor neuron derivation was limited tocultures passaged at about day 11 of differentiation suggesting reducedpatterning response at very high cell densities as observed for hESCderived R-NSCs (Elkabetz, et al., Genes Dev 22 (2), 152 (2008)). Thesedata demonstrate robust patterning response in Noggin/SB431542 treatedneural progeny and derivation of relevant neuron subtypes after shortdifferentiation periods (approximately 19 days) compared to 30-50 dayswhen using stromal feeder mediated induction protocols (Lee, et al.,Stem Cells 25 (8), 1931 (2007); Tomishima, et al., Stem Cells 25 (1), 39(2007)).

As an alternative to the specific Smad inhibitors used here, it ispossible to block both distinct Smad pathways using alternativeinhibitors or mechanisms. Dorsomorphin is a small molecule alternativeto Noggin, targeting the same pathway. Concentrations ranged from 10 uMto 30 nM, each individual amount added to 10 uM of SB431542. Theefficiency was not as high as used with Noggin/SB431542 based on thepercentage of PAX6+ cells, but the ratios a combination of Noggin anddorsomorphin with 0431542 allowed for a 15 fold reduction in theconcentration of Noggin necessary to obtain equivalent efficiency andcell viability. The small molecule was more cost efficient than Noggin,and the reduction of price is desirable. It is possible to utilize othermolecules as well, although there is currently no known alternativesmall molecule to SB431542 that blocks the entire range of targets, butthis example demonstrates that total Smad cloaked (the two knowpathways) can result in robust and synergistic effects that yield ahighly homogenous population of neural cells. These alternative methodscould also include Smad blockade through mechanism including interferingDNA (to include antisense, siRNA, shRNBA, and standard methods known tothe art), or overexpression of a protein that can block, compete orotherwise present Smad 4 function (such as overexpression of Smad 7).

Specific cell fates were tested for their ability to survive, migrate,and function as desired in mammals. The inventors can transplantneurogenic tissue from hiPSCs that are differentiated using Noggin andSB431542 protocol followed by a dopamine neuron induction protocol aretransplanted into the brains of recipient mice (specifically, Nod/SCID)and assessment of the engraftment potential of the cells is assessed.

Further, it is possible to observe pigmented cells when the cells arefurther differentiated from the Noggin and SB431542 protocol towardsmore mature neurons (both motor neurons and dopamine neurons). This datasuggests that both melanocytes and retinal-pigmented epithelium arebeing produced. Additionally, PAX6+ central nervous system progenitorcells and a PAX6-HNK1+ peripheral nervous system progenitor cell wereobserved. Recent publications have reported the reprogramming of humansomatic cells into induced pluripotency stem cells (hiPSCs) (Takahashi,et al., Cell 131 (5), 861 (2007); Suter, et al., Stem Cells 27, 49-58(2008)). Next it was determined if dual SMAD inhibition could be used toreliably generate a broad repertoire of hiPSC derived neural cell types.Given the expected intrinsic variability among hiPSC clones,reproducible differentiation results would confirm the robustness of ournovel differentiation protocol. Two hiPS clones (IPS^(C14), IPS^(C17);FIGS. 3a-i and a -ii) were generated using lentiviral transduction ofhuman fetal lung fibroblasts with cMYC, KLF4, OCT4, and SOX2. Bothclones express the pluripotency markers including Nanog, Tra-1-60, andSSEA-3 at the undifferentiated state and are capable of differentiatinginto derivatives of the three germ layers. Upon neural induction via thenoggin/SB431542 protocol, both clones yielded nearly homogenouspopulations of PAX6+ cells by day 11 of differentiation (FIGS. 3 b-i,b-ii). Using the strategies described above manipulating, cell density,passage, and patterning factors both hiPSC clones could be readilybiased towards generating HNK1+ putative neural crest progeny (Figurec-i,c-ii), hiPSC derived R-NSCs (FIG. 3d -i,d-ii), and specific hiPSCderived neuron subtypes including somatic motor neurons (FIG. 3e-i,e-ii) and dopamine neurons (FIG. 3f -i,f-ii). These data demonstraterobustness and modularity of the dual SMAD inhibition strategy beyondhESC differentiation. The novel protocol offers an efficient, defined,and robust platform for the rapid generation of hiPSC derived neuralcell types.

Thus a novel method of neural differentiation was discovered bycombining at least two signaling inhibitors, i.e. SB431542 and Noggin.While for most of the studies presented herein used a 11-day treatmentperiod, subsequent studies showed that comparable levels of neuralinduction were achieved when the treatment is shortened to the first 5days of differentiation (FIG. 10). This reduced time of treatment shouldfurther reduce complexity and cost, particularly in the case ofrecombinant Noggin. In some embodiments, an inhibitor of SMAD signalingis replaced by an inhibitor of a Bone morphogenetic protein (BMPsignaling) pathway. Small molecule inhibitors of the BMP pathway arealso available that could potentially substitute for noggin function andfurther reduce costs. Thus, in some embodiments, noggin is replaced byDorshomophin. In other embodiments, noggin is replaced by LDN-193189,for an exemplary structure see

(another example, ‘Stemolecule™ BMP Inhibitor LDN-193189’ StemGent,Cambridge, Mass.).

Cranial placodes are transient developmental structures critical for theformation of the lens, nasal epithelium, otic structures, cells of theadenohypophysis, and multiple cranial nerves including the trigeminalganglion. Little is known about human placode biology due to theinaccessibility of the tissue during development and the lack ofvalidated markers. Most of our knowledge is extrapolated from otherspecies such as xenopus, zebrafish, chick, and to lesser extent mousedevelopment. Here, The inventors report the derivation of cranialplacodes and placode derived sensory neurons from human embryonic stemcells (hESCs). Six1+ hESC derived placode precursors are obtained athigh yield (71% of total cells) within 11 days of differentiation usinga modified dual SMAD inhibitor protocol. Six1+ cells co-express otherputative placode markers such as eyes absent homolog 1 (Drosophila)(Eya1), Dachshund homolog 1 (Dach1), eyes absent homolog 4 (Drosophila)(Eya4), and SIX homeobox 3 (Six3; sine oculis homeobox homolog 3(Drosophila)) and temporal transcriptome analysis identifies additionalplacode and sub-placode specific markers. Prospective pan-placodeprecursor cells were isolated based on the expression of p75 in theabsence of HNK1 expression. Specific enhancer GFP constructs enablemarking placodal cells with putative specificity to a subset of placodalregions. Human ESC derived placodal cells were highly efficientlyconverted into pure populations of sensory neurons expressing insulingene enhancer protein ISL-1 (Isl1), brain-specific homeobox/POU domainprotein 3A (Bm3a), β-III-tubulin (Tuj1) and peripherin. The isolation ofhESC-derived placodal represents a novel model system to study humanplacode development and enable the derivation of unlimited numbers ofpreviously inaccessible sensory neuron population for the study ofsensory function and pain.

Cranial placodes are transient developmental structures that give riseto the peripheral olfactory system, the lens, the anterior pituitary,otic structures, and sensory ganglia including trigeminal neurons.Defects in placode development are involved in a range of humancongenital malformations, including blindness, deafness and loss of thesense of smell (Baker, et al., Dev Biol, 232(1):1-61 (2001); Bailey, etal., Curr Top Dev Biol, 72:167-204 (2001), herein incorporated byreference). Cranial placode development has been well characterized invarious model organisms including Xenopus, chick and zebrafish (Baker,et al., Dev Biol, 232(1):1-61 (2001); Bailey, et al., Curr Top Dev Biol,72:167-204 (2006); Bhattacharyya, et al., Curr Opin Genet Dev. 14(5):520-6 (2004); and Baker, et al., Development, 2000. 127(14): p. 3045-56,all of which are herein incorporated by reference). Despite theimportance of placode biology in development and disease, however, humanplacode development has remained unexplored. This is largely due toinaccessibility of early human placode tissue and the associated lack ofappropriate markers and techniques.

Embryonic stem cells have the unique ability to self-renew in a nearlyunlimited fashion while retaining the ability to differentiate into allthe various cell types that make up an adult organism. During humandevelopment, pluripotent cells of the inner cell mass (ICM) and epiblastfrom which human ES cells are derived gives rise to the three germlayers and all subsequent derivatives, including placode cells. One keyquestion is whether the in vivo differentiation potential of the humanICM were harnessed using human ES cell-based culture systems in vitro.

Over the last few years a number of protocols have been developed by ourlab and others for the directed differentiation of human ES cells intovarious tissue specific cell types, such as midbrain dopamine neurons,Perrier, et al., Proc Natl Acad Sci USA, 101(34):12543-8 (2004), hereinincorporated by reference, spinal motoneurons Li, et al., NatBiotechnol, 23(2): 215-21 (2005), herein incorporated by reference,multipotent mesenchymal precursors, Barberi, et al., PLoS Med, 2(6):e161(2005); Barbed, et al., Nat Med, 2007. 13(5):642-8, herein incorporatedby reference, cardiac cells, Laflamme, et al., Nat Biotechnol,25(9):1015-24 (2007), herein incorporated by reference, andhepatocyte-like cells Agarwal, et al., Stem Cells, 26(5):1117-27 (2008),herein incorporated by reference. Directed differentiation into cells ofperipheral neuron identity has been achieved via a neural crestprecursor intermediate. The initial protocols on generating human EScell-derived neural crest cells, Lee, et al., Nat Biotechnol,25(12):1468-75 (2007), herein incorporated by reference were based on aMS5 co-culture system promoting neural induction, Perrier, et al., ProcNatl Acad Sci USA, 101(34):12543-8 (2004); Barbed, et al., NatBiotechnol, 21(10):1200-7 (2003), herein incorporated by reference. TheMS5 culture system was used successfully for deriving and isolatingvarious neural crest fates from human. ES cells and human IPS cells, andfor modeling a familial dysautonomia (FD), a rare human genetic disorderaffecting neural crest-derived neurons, Lee, et al., Nature,461(7262):402-6 (Epub 2009 Aug. 19) herein incorporated by reference.

Recently, our lab developed a novel and defined neural inductionstrategy that is based on the concomitant inhibition of the BMP andTGFb/Activin/Nodal signaling pathways, Chambers, et al., Nat Biotechnol,27(3):275-80 (2009), herein incorporated by reference. Exposure toNoggin (N) and SB431542 (SB) leads to a synchronized and rapiddifferentiation of human ES cells or IPS cells towards neural fatesunder adherent culture conditions and therefore obviates the need forboth co-culture and embryoid body formation during the inductionprocess. The more rapid and synchronized differentiation response usingthe N-SB protocol enables testing of the precise relationship ofspecific morphogens in biasing developmental fate in vitro.

Here The inventors describe a modified N-SB protocol that allows theefficient derivation of highly enriched populations of placodalprecursors. Placodal fate is induced at the expense of neuroectodermalcells upon withdrawal of noggin treatment 48 hours after N-SB induction.These data illustrated the use of the N-SB induction system to optimizethe generation of non-CNS derivatives, demonstrate the importance ofendogenous BMP signaling during hESC differentiation and enable thederivation of unlimited numbers of placode derivates such as cranialsensory neurons for the study of sensory function and pain.

The inventors made several observations from the studies describedherein which are presented as follows for use in methods of the presentinventions.

BMP Dependent Specification of Placodal Fates During HESCDifferentiation.

BMPs exert wide-ranging effects on early embryonic fate specification invivo and are involved in the specification of various extra-embryonicstructures, determination of definitive mesodermal cells, specificationof non-neural ectoderm, placode and neural crest tissues. The use of theN-SB culture system enables a highly synchronized and efficientdifferentiation of human ES cells. In our current study the N-SB systemreveals an exquisite control of BMP dose and timing of application inthe specification of placodal fates. Important questions remain whetherthe system were used similarly to optimize differentiation towardsnon-neural ectoderm fates and the generation of primitive skin precursorcells. Data indicated that a subset of Six1 negative cells under themodified N-SB culture conditions express p63, a known marker of earlyepidermal precursors during development.

The Emergence of Putative Pan-Placodal Fates During Human Embryonic Stem(ES) Cell Differentiation.

Highly efficient differentiation towards Six1+ fates and the rapidemergence of insulin gene enhancer protein ISL-1, also known as ISL LIMhomeobox 1, (Isl1)+ cells during human ES cell differentiation suggestthat hESC derived cells may initially adopt a pre-placodal precursorfate. The emergence of a preplacodal region has been described duringxenopus and zebrafish development marking a horseshoe shaped area in themost anterior region of the embryo surrounding the anteriorneuroectodermal cells. The current study focused primarily on thegeneration of sensory neuron precursors from the Six1+ placodal regions.However, future studies should address the plasticity of these placodalcells upon exposure to alternative differentiation regimens. Ofparticular interest may be the derivation of adenohypophyseal cells tostudy specification of various hormone producing cell types. Such cellsare of interest for developmental studies and for studies aimed atdefining pharmacological control of hormone release. Approximately, 15%of the cells at the Six1+ stage express Lhx3, a marker ofadenohypophyseal precursor cells. Expression of CGA, the precursorprotein in the production of adenohypophyseal hormones, was observedduring human ES cell differentiation in the modified N-SB protocol atday 11 of differentiation (FIG. 12D). The presence of Lhx3+ putativeadenohypophyseal precursor cells and the expression of CGA showed thatcells of adenohypophyseal lineage were readily induced using themodified N-SB protocol.

Sensory Neuron Specification From Six1+ Placodal Precursors.

Our findings strongly indicate a placodal origin of the sensory neuronpopulations generated in the modified N-SB protocol. This is based onthe expression of Six1 in the precursor clusters isolated for subsequentsensory neuron generation and the co-expression of Six-1 in early stagesensory neurons. Placode derived sensory neurons share various markerswith sensory neurons derived from neural crest lineages such as Brn3A,Isl1 and Peripherin. However, the modified N-SB protocol shows highlyefficient induction of FoxG1B and other anterior markers expressed inplacodal precursor and not expressed in early neural crest lineages. Itwill be interesting to explore how initial cell density (Chambers etal., Nature Biotechnology 27(3) 275-280, 2009, Corrigendum: in NatureBiotechnology 27(4):1) and modulation of Wnt signaling (reference) mayenable specification of placodal versus neural crest derived sensoryneuron populations. Access to highly purified populations of cranialsensory neurons represent a novel tool for the future development highthroughput drug discovery assays. For example compounds modulatingplacode derived trigeminal neurons may be of particular interest giventhe well known clinical syndromes associated with trigeminal nervedysfunction.

I. Specification of Functional Floor Plate Tissue from Human EmbryonicStem Cells Occurs at the Expense of Anterior Neurectoderm.

The floor plate (FP) is a critical signaling center during neuraldevelopment located along the ventral midline of the embryo. Little isknown about FP development in humans, due the lack of tissueaccessibility. This disclosure describes the derivation of humanembryonic stem cells (hESC-) and subsequently derived FP tissue capableof secreting Netrin-1 and SHH and influencing patterning of primary andhESC derived tissues. Induction of FP in hESCs is dependent on early SHHexposure and occurs at the expense of anterior neurectoderm (AN). Globalgene expression and functional studies identify SHH-mediated inhibitionof DKK-1 as key factor in AN repression. hESC derived FP tissue is shownto be of anterior SIX6+ character but responsive to caudalizing factorssuppressing SIX6 expression and inducing a shift in expression ofregion-specific SHH enhancers. These data established hESC derived FP asan experimental model system and define early signaling events thatmodulate FP versus AN specification.

Neural development is dictated in time and space by a complex set ofsignals that instruct neural precursor identity. While significantprogress has been made in animal models, human neural developmentremains much less understood. Human embryonic stem cells (hESCs) offeran accessible and manipulatable cell platform to model the early stagesof human development.

Previous studies have reported the directed differentiation of mouse(Wichterle et al., 2002; Barberi et al., 2003; Watanabe et al., 2005)and human (Perrier et al., 2004; Li et al., 2008; Eiraku et al., 2008)ESCs into specific neuron types in response to patterning factorsdefining anterior/posterior (A/P) and dorso-/ventral (D/V) CNS identityThese studies demonstrate evolutionary conservation of signaling systemsthat specify the major CNS regions. In mammals, sonic hedgehog (SHH) isthe key ventralizing factor acting in a dose-dependent manner to specifythe various ventral cell types including cells expressing floor plate(FP) in primary neural explants (Briscoe and Ericson, 1999) and in mouseES cells (Mizuseki et al., 2003). While application of SHH tohESC-derived neural cells has been shown to induce various ventralneuron types, the derivation of floor plate (FP) tissue itself has notyet been reported. As FP is one of the most important signaling centers,the ability to produce FP from human ES cells will be a major stepforward in furthering our studies of early human neural development.

The FP runs along the most medial aspect of the ventral neural tubeextending most caudally from the spinal cord, through the midbrain, upto the diencephalon with its anterior limit being just below the zonalimitans intrathalamica (lessen et al., 1989). Interestingly, at themost anterior aspect the FP stops where the anterior neurectoderm (AN)begins, and studies have shown that AN commitment renders cellsincapable of responding to FP inductive signals (Placzek et al., 2003).Classic studies have shown FP cells to exhibit a unique, flatmorphology, and to express FP specific markers including SHH, FOXA2,F-Spondin, and Netrin-1 (Placzek, 1995). Studies in mouse and chickembryos have identified two major organizer functions for the FP: thesecretion of the morphogen SHH patterning the ventral neural tube(Placzek and Briscoe, 2005), and the expression of Netrin-1 guidingcommissural axons across the midline (Charron et al., 2003). The FP isgenerally considered a non-neurogenic region. However, genetic lineagemapping studies in the mouse have recently reported that the midbrain FPselectively exhibits neurogenic potential and is the source of ventralmidbrain dopamine neurons (Kittappa et al, 2007; Ono et al., 2007;Joksimovic et al., 2009).

To date, little is known about FP development in humans, due to lack ofaccessibility to tissue. In animals, the FP is a major site of SHHproduction and several human developmental disorders are related toalterations in midline SHH signaling (Mullor et al., 2002) includingcertain forms of holoprosencephaly and microphthalmia, skeletaldisorders including various cleft plate syndromes, and tumor conditionssuch as Gorlin's syndrome; a rare genetic disorder caused by a mutationin the SHH receptor Patched 1. Thus, understanding how human FP isgenerated will be critical for comparative developmental studies ofhuman neural patterning and axonal pathfinding and the resulting cellscould potentially serve as a source of specific neuron types that have aFP origin.

The Examples provided herein demonstrate exemplary directeddifferentiation of hESCs into FP tissue, as the first example ofgenerating a human developmental organizer structure in vitro. Theinventors showed that human FP specification is dependent on earlyhigh-dose SETH signaling that represses DKK1-mediated specification ofAN. Functionality of the FP is demonstrated by secretion of Netrin-1 andSHH and the ability to induce ectopic FP tissue and neurite outgrowth inprimary mouse and rat explants.

Human ESC derived FP adopts anterior identity by default but werespecified to posterior fates in response to caudalizing cues providingaccess to region-specific FP tissue. The experimental system presentedhere, and in combination with compositions and methods described forhighly efficient neural conversion of human ES and iPS cells by dualinhibition of SMAD signaling, Nat. Biotechnol. 26, 275-280 (2009);published online 1 Mar. 2009; corrected after print 16 Mar. 2009,Corrigendum: Chambers, et al., should facilitate studies on FP-mediatedsignaling events critical during early human neural development.

II. Stem Cell Lines

The present invention is not limited to the use of any particular typeof human stem cells. Indeed, the use of a variety of types of human stemcells is contemplated. Methods for obtaining totipotent or pluripotentcells from humans, monkeys, mice, rats, pigs, cattle and sheep have beenpreviously described. See, e.g., U.S. Pat. Nos. 5,453,357; 5,523,226;5,589,376; 5,340,740; and 5,166,065 (all of which are specificallyincorporated herein by reference); as well as, Evans, et al.,Theriogenology 33(1):125-128, 1990; Evans, et al., Theriogenology33(1):125-128, 1990; Notarianni, et al., J. Reprod. Feral.41(Suppl.):51-56, 1990; Giles, et al., Mol. Reprod. Dev. 36:130-138,1993; Graves, et al., Mol. Reprod. Dev. 36:424-433, 1993; Sukoyan, etal., Mol. Reprod. Dev. 33:418-431, 1992; Sukoyan, et al., Mol. Reprod.Dev. 36:148-158, 1993; Iannaccone, et al., Dev. Biol. 163:288-292, 1994;Evans & Kaufman, Nature 292:154-156, 1981; Martin, Proc Natl Acad SciUSA 78:7634-7638, 1981; Doetschman et al. Dev Biol 127:224-227, 1988);Giles et al. Mol Reprod Dev 36:130-138, 1993; Graves & Moreadith, MolReprod Dev 36:424-433, 1993 and Bradley, et al., Nature 309:255-256,1984.

In preferred embodiments, undifferentiated human embryonic cells linesare contemplated for use, for examples, cell line WA09, and the like.

III. Discussion

An important finding of the studies described herein, is the “default”nature and anterior bias of the embryonic cell derived FP tissue. Thelack of any obvious mesodermal intermediates in both NSB (Chambers etal., 2009; FIG. 4) and the FP (FIG. 4) induction protocol presentedhere, suggests that the in vitro derivation of neuroectoderm and FPtissue is not dependent on any additional mesoderm derived signals.Interestingly, FP induction occurs readily even in the presence ofSB431542, an inhibitor of TGFb/Activin/Nodal signaling in contrast todata in zebrafish where nodal is thought to be essential for FPinduction (reviewed Placzek and Briscoe, 2005). The anterior default ofthe FP tissue reported here is reminiscent of the anterior defaultobserved in hESC derived neuroectodermal cells. Studies in chickdevelopment have proposed two distinct origins of anterior versusposterior FP namely progenitors in the epiblast and axial mesoderm(Placzek et al., 2003). This data indicated that human FP precursorcells, similar to neuroectodermal cells, are capable of beingrespecified towards posterior FP identity in response to caudalizingfactors including FGF8 and Wnt-1.

To date, it has not been clearly shown whether expression of AN markersinhibits the ability of cells to yield certain lineages. In hESC derivedneural rosette cells, expression of BF1 does not preclude patterningtowards posterior CNS fates including HB9+ somatic motoneurons.

However, the efficiency of generating caudal neuron fates issignificantly reduced as compared to BF1-negative rosettes (Elkabetz, etal., 2008). Previous studies in primary mouse explants showed that ANcells are not competent to differentiate into FP cells in response toSHH alone (Placzek, et al., 1993). The methods and results describedherein during the development of the present inventions showed an ANcommitment that was not capable of FP specification.

An additional key finding described herein is a dramatic (strong)induction of DKK-1 during NSB-mediated neural differentiation. Duringneural differentiation of mouse ESCs exposed to extrinsic DKK-1 enhancedAN induction under serum-free embryoid body (SFEB) conditions (Watanabeet al., 2005). Thus the inventors contemplate that early induction ofendogenous DKK-1 during neural differentiation is least in partresponsible for the AN default phenotype observed in hESCs. Functionalstudies demonstrated herein that inhibition of DKK-1 using blockingantibodies significantly improved FP yield. Further, addition of DKK-1antibodies along with SHH at day 5 or later time-points was notsufficient to extend the temporal window of FP competency (FIGS. 5H and5I). Similarly, the addition of WNTs (Wnt-1, Wnt3A or GSK inhibitor(BIO)) to neural rosette stage cells was not sufficient to induced FPcompetency (FIG. 11). These data demonstrated that very early DKK1mediated AN bias suppresses FP potential of hESC derived precursors.

Additional data in BF1 knockdown hESC lines showed enhanced FP yieldwith improved yield of posterior CNS cell types such as HB9+motoneurons. Thus a model system presented herein is contemplated to besuitable to assess the contribution of other molecules with WNTinhibitory function such as Cerberus (Bouwmeester, et al., 1996) to ANspecification and repression of FP fate. Finally, the system and methodsdescribed herein are contemplated to allow the identification ofpotential upstream regulators of DKK-1 expression regulating AN defaultin hESCs.

Suppression of DKK-1 and subsequently AN fates via early exposure tohigh levels of extrinsic SHH results was a surprising result, as SHH isa classic ventralizing factor and not known to exert effects on APspecification during neural development. Our studies did not addresswhether regulation of DKK-1 by SHH is direct or caused by inducing analternative precursor population devoid of DKK-1 expression. While DKK-1inhibited FP induction, exposure to WNT1 enhanced the derivation of FPtissue from hESCs. These data raised the question whether RA that inducea similar increase in FP yield would affect WNT signaling or whetherinduction of posterior fates enhances FP yield independently of Wntsignaling.

The availability of unlimited number of FP cells of defined regionalidentity provides a valuable tool for studying human neural development.Recent studies in the mouse suggest that some regions of the FP, beyondkey roles in neural patterning and axonal path finding, may serve as asource of specific neuron types including midbrain dopamine neurons. Theinventors demonstrated re-specification of regional identity of hESCderived FP towards midbrain character based on the expression ofmidbrain specific markers and the activation of midbrain specific SHHenhancer elements. The inventors found evidence that hESC derived FPtissue is capable of yielding TH+/FOXA2+ putative midbrain DA neurons(FIG. 11). The results shown herein provided insights into the inductionand regional specification of human FP versus AN fates and establishedhESCs as a powerful model system to create a functional organizer tissuesuitable for modeling more complex interactions during humandevelopment.

In conclusion, exemplary data shown herein showed that neuraldifferentiation hESCs default towards an AN fate by upregulating DKK-1and subsequently BF1, while AN commitment actively repressed FPcompetency in hESC progeny. However, an early high level of SHH reducedDKK-1 levels enabling FP induction at the expense of AN whileloss-of-function of DKK-1 or BF1 increased FP production.

to caudalizing factors. This is summarized in FIG. 6E.

Thus human ESC derived FP is anterior by default but was posteriorizedin response

TABLE A Exemplary ranges of amounts of compounds for obtaining neuralcells of the present inventions. Concentration ConcentrationConcentration Concentration range of range of range of Sonic range ofNoggin Dorsomorphin SB431542 C25II (SHH) Noggin with 125-500 NA 0.001 to1000 NA SB431542 ng/mL microM Noggin with 500 ng/mL NA 0.001 to 1000200-2000 SB431542 and microM ng/mL SHH Dorsomorphin NA 100-5000 nM,0.001 to 1000 NA with best results 600 nM microM SB431542 Noggin with25-500 ng/mL, 100-5000 nM, 0.001 to 1000 NA Dorsomorphin high efficiencybest results 600 microM with to 30 ng/mL nM SB431542

Thus, numerous embodiments of the present inventions are summarized inthe following Tables.

TABLE B Exemplary time of addition of compounds of the presentinventions for producing neural cell types of the present inventions.Start Cell Type Stem cells Stem cells Stem cells Modified N-SB N-SB andconditions including iPS including iPS and including iPS and treatedhESC or treated and hESC. hESC. hESC. iPS: KSR medium hESC or Lowdensity of High density of Low or high or conditioned iPS in cells: KSRcells: KSR medium density of cells: medium non- medium or or conditionedKSR medium or adherent conditioned medium conditioned embryoid mediummedium bodies N-SB: Noggin Add both day 0 Add both day 0 of Add both day0 of NA Add both and/or of culture of culture and culture and day 0 ofDorsomorphin culture and continue adding continue adding culture withSB431542 continue adding fresh aliquots when fresh aliquots and freshaliquots feeding cells when feeding cells continue when feeding addingcells fresh aliquots when feeding cells; SB withdraw at or around Day 7Modified N-SB NA NA NA Add both day 0 of NA Noggin/ culture and replaceDorsomorphin with cell media withdrawal 2 days without Noggin (1-3)after N-SB and/or induction Dorsomorphin day 1 (ranging from 6 hours to4 days after day 0) SHH or C25II NA NA Add day 1 after N- NA NA SBadditions (ranging 0-5 days) Gradually replacing KSR media with N2 mediabetween Day 5 and 11. Resulting cells CNS progenitor CNS progenitorFOXA2+ (BF1 Six1+ placodal High cells (PAX6+) cells (PAX6+) (R- reduced)SOX17- precursors leading efficiency and PNS NS cells and neural cellsto motor progenitor cells patternable i.e. FP Brn3a+ progenitor neuron(p75+, HNK-1+) neuronal differentiation cells, leading to cellspopulations of with FP anterior immature neuronal motoneurons and cellsas a default cells, Tuj1+, dopaminergic type but posterior peripherin+and neurons within FP tissue can be mature neurons. 19 d of initiatinginduced in the differentiation) presence of caudalizing factors such asWnt-1, FGFF8 or RA.

REFERENCES

The following references are herein incorporated in their entirety.Barberi, et al., (2003). Nat Biotechnol. 10:1200-1207; Bouwmeester, etal., (1996). Nature 382:595-601; Briscoe, J., and Ericson, J. (1999).Semin Cell Dev Biol. 3:353-62; Chambers, et al., (2009). Nat Biotechnol27, 275-280; Charrier, et al., (2002). Development 129:4785-4796;Charron, et al., (2003). Cell 113:11-23; D'Amour, et al., (2005). NatBiotechnol 23, 1534-1541; Dennis, et al., (2003). DAVID: Database forAnnotation, Visualization, and Integrated Discovery. Genome Biol 4, P3;Eiraku, et al., (2008). Cell Stem Cell 3, 519-53; Elkabetz, et al.,(2008). Genes Dev 22:152-165; Ericson, et al., (1996). Cell 87, 661-673;Fasano, et al., (2007). Cell Stem Cell 1, 87-99; Fasano, et at, (2009).Genes Dev 23, 561-574; Glinka, et al., (1998). Nature 391, 357-362;Haung, et at, (2009). Nat Protoc 4, 44-57; Hunter, et al., (1991). ProcNatl Acad Sci USA 88, 3666-3670; Ivanova, et al., (2006). Nature 442,533-538; Jeong, et al., (2003). Development 130, 3891-3902; Jeong, etal., (2005). Development. 133, 7761-7772; Jeong, et al., (2008) NatGenet 40, 1348-1353; Jessell, (2000). Nat Rev Genet 1, 20-29; Jessell,et al., (1989). Ciba Found Symp 144, 255-276; discussion 276-280,290-255; Joksimovic, et al., (2009). Nat Neurosci 12, 125-131;Kimura-Yoshida, et al., (2006). PNAS 104, 5919-59249; Kittappa, et al.,(2007). PLoS Biol 5, e325; Li, et al., (2008). Stem Cells 4, 886-89399;Lois, et al., (2002). Science 295, 868-872; Lyuksyutova, et al., (2003).Science 302, 1903-1904; Matise, et al., (1998). Development 125,2759-2770; Mizuseki, et al., (2003). Proc Natl Acad Sci USA 100,5828-5833; Mukhopadhyay, et al., (2001). Dev Cell 3, 423-434; Mullor, etal., (2002). Trends Cell Biol 12, 562-569; Ono, et al., (2007).Development 134, 3213-3225; Perrier, et al., (2004). Proc Natl Acad SciUSA 101, 12543-12548; Placzek, et at, (1993). Development 117, 205-218;Placzek, M. (1995). Curr Opin Genet Dev 5, 499-506; Placzek, et al.,(2003). Development 130, 4809-4821; Placzek, et al., (2005). Nat RevNeurosci 6, 230-240; Roelink, et al., (1994). Cell 76, 761-775; Shen, etal., (2006). Nat Neurosci 9, 743-751; Shirasaki, et al., (1995). Neuron14, 961-972; Suter, et al., Stem Cells, 27(1):49-58 (2009); Venezia, etal., (2003). PLoS Biol 10, e301; Watanabe, et al., (2005). Nat Neuro 3,288-296; Weinstein, et al., (1999). Annu Rev Cell Dev Biol 15, 411-433;Wichterle, et al., (2002). Cell 110, 385-397; Zhang, et al., NatureBiotechnology 19, 1129-1133 (2001); and Zoltewicz, et al., (1999).Development 126, 5085-5095.

EXPERIMENTAL

The following examples serve to illustrate certain embodiments andaspects of the present invention and are not to be construed as limitingthe scope thereof. In the experimental disclosures which follow, thefollowing abbreviations apply: N (normal); M (molar); mM (millimolar);μM (micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); pmol (picomoles); g (grams); mg (milligrams); μg(micrograms); ng (nanograms); pg (picograms); L and (liters); ml(milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm(micrometers); nm (nanometers); U (units); min (minute); s and sec(second); deg (degree); and ° C. (degrees Centigrade/Celsius).

The following are general cell culture formulations.

hESC Medium for Maintenance (1 Liter):

800 mL DMEM/F12, 200 mL of Knockout Serum Replacement, 5 mL of 200 mML-Glutamine, 5 mL of Pen/Strep, 10 mL of 10 mM MEM minimum non-essentialamino acids solution, 1000 μL of β-mercaptoethanol, bFGF (finalconcentration is 4 ng/mL)

KSR Medium for hESC Differentiation (1 Liter):

820 mL of Knock out DMEM, 150 mL of Knock out Serum Replacement, 10 mLof 200 mM L-Glutamine, 10 mL of Pen/Strep, 10 mL of 10 mM MEM, 1 mL ofβ-mercaptoethanol

N2 Medium for hESC Differentiation (1 Liter):

985 ml dist. H₂O with DMEM/F12 powder, 1.55 g Glucose, 2.00 g NaHCO₃, 25mg insulin, 0.1 g apotransferrin, 30 nM sodium selenite, 100 μMputrescine, 20 nM progesterone

DMEM with 10% FBS for Preparing PMEF (1 Liter):

885 mL of DMEM, 100 mL of FBS, 10 mL of Pen/Strep, 5 mL of L-Glutamine

Alpha MEM with 10% FBS for preparing MS-5 feeder (1 liter):

890 mL of Alpha MEM, 100 mL of FBS, 10 mL of Pen/Step

Gelatin Solution (500 ml):

Dissolve 0.5 g of gelatin in 500 ml of warm (50-60° C.) Milli-Q water.Cool to room temperature.

Noggin was purchased from R&D system as Catalog Number: 719-NG,

Recombinant Mouse Noggin Fc Chimera.

Example I Bone Morphogenetic Protein (BMP) Levels Determine Placode FateIdentity.

Sensory placodes are developmental structures formed at the interface ofearly neuroectodermal and non-neural ectoderm tissue. To address whetherthe N-SB culture system is suitable for the derivation of placodalcells, The inventors first established a set of markers to identifyplacode identity in human embryonic stem cells (hESCs)-derived cultures.From studies in other model organisms, The inventors postulated a numberof candidate markers to identify human placodal precursor during hESCdifferentiation including member of the Six, Eya, and Dlx family oftranscription factors. Six1 marks the pre-placodal region and placodalcells in model organisms but is also expressed in skeletal muscleprecursors. Immunocytochemical analyses revealed 6%±4% Six1+ cells atday 11 of N-SB differentiation. The absence of the expression ofskeletal muscle markers in Six1+ cells suggested placodal precursor cellidentity.

A large number of developmental studies have demonstrated a criticalrole for BMP signaling during early ectodermal patterning in vivo. Onemodel suggests that a gradient of Bmp activity within the ectodermallocates different cell fates, with high levels of signaling promotingepidermis, moderate levels inducing placodes, intermediate levelsspecifying neural crest and complete absence of Bmp activity beingrequired for neural plate formation in vivo Streit, et al., Dev Biol,2004. 276(1): p. 1-15, herein incorporated by reference. To test whetheraddition of exogenous BMPs enhances the derivation of Six1+ cells, Theinventors exposed SB431542 (1 μM) treated hESCs to various concentrationof BMP4. However, early addition of BMP4 caused a dramatic morphologicalchance of the cells and induction of cdx2 suggesting differentiationtowards trophectodermal fates. In agreement with our findings previousstudies in the absence of SB431542 have demonstrated that early exposureto BMPs can drive trophectodermal fates during hESC differentiationChambers, et al., Nat Biotechnol, 2009. 27(3): p. 275-80; Xu, et al.,Nat Biotechnol, 2002. 20(12): p. 1261-4, herein incorporated byreference.

We next explored whether simple withdrawal of the BMP inhibitor nogginduring N-SB differentiation may enhance the emergence of placodal fatesby de-repressing endogenous BMP signaling. To this end The inventorsperformed a time course study-removing noggin at different time pointsof the NSB protocol (FIG. 11A) while monitoring the induction ofplacodal marker (FIG. 11B) trophectodermal, neuroectodermal by qRT-PCRanalysis at day 11 of differentiation. The inventors observed thatwithdrawal of noggin at day 2 or 3 of differentiation yielded efficientinduction of Six1 while noggin withdrawal at day 1 of differentiationlead to the induction of Eya1 in the absence of Six1 expression. Theinduction of morphological changes and expression of Cdx2 in culturessubjected to day 1 noggin withdrawal indicated differentiation towardstrophectodermal fates and suggested that Eya1 is expressed introphectodermal precursors in addition to placodal cells.Immunocytochemical analysis demonstrated that that noggin withdrawal atday 3 of differentiation induced a switch from primarily Pax6+neuroectodermal cells obtained under standard N-SB conditions topopulations composed of 71% Six1+ putative placode precursor cells (FIG.11C, D). Co-labeling studies demonstrated co-expression of otherplacodal markers in Six1+ cells such as Eya1 in the absence of markersof skeletal muscle fates.

Microarray analysis reveals novel human placode progenitor geneexpression: To obtain an unbiased measure of placode induction ofplacode precursor cell identity The inventors performed a time courseanalysis of global gene expression using the Illumina bead arrayplatform. RNA was collected at five time points during differentiation(Day 1, 3, 5, 7, and 11) in control N-SB cultures (yielding anteriorneural plate cells; noggin/SB431542 treatment for day 1-11) and underconditions promoting placodal fates (anterior placode cells; SB431542treatment for 11 days; Noggin treatment from day 1-3). Prior themicroarray analysis the quality of each sample was verified forexpression of a panel of placode markers (Six1, Dlx3, Eya1) and theabsence of other lineages such as Foxa2 (endoderm), Sox17 (endoderm),MyoD (skeletal muscle), cdx2 (trophoblast), and T (Brachyury)(mesoderm).Global gene expression studies were carried out in three independentsamples for each time point and culture condition. Data were convertedinto log 2 ratios comparing levels of gene expression in placode versusN-SB protocol during differentiation (FIG. 12A-D).

The time course data were subjected to gene ontology (GO) enrichmentanalysis using DAVID (http://david.abcc.ncifcrf.gov/; Dennis, et al.,Genome Biology 2003 4:P3, 2003. 4(5): p. P3, herein incorporated byreference) as unbiased assessment of placode transcription profile.Among the transcripts highly enriched in placode conditions versus NSBcontrol cultures at day 5 and day 7 of differentiation were genesassociated with the sensory organ development, BMP and Wnt pathways,inner ear development and neural patterning. Enrichment for sensoryorgan development and neural patterning factors further confirm anteriorplacode identity of cultures derived using the modified N-SB protocol.Neural plate markers and neuronal markers were also down-regulated inthe modified N-SB (noggin withdrawal after 2 days of differentiation)versus NSB (i.e. treatment of cells with at least 2 SMAD or BMPsignaling compounds) protocol.

To gain insight into specific genes differentially expressed duringplacode specification, The inventors performed pair-wise comparisons atfor each differentiation stage. While the majority of genessignificantly regulated at day 5 and day 7 of differentiation (ascompared to day 1) were shared in NSB and modified N-SB protocol, asubset of transcripts was differentially regulated. In particular, Theinventors noticed an increase in Islet-1, a very well known marker forsensory neuron development, in the modified N-SB protocol. Isl1 is alsoexpressed in other lineages such as motoneuron, heart progenitors andpancreatic islet cells. The early expression onset of expression duringhESC differentiation suggests that Islet-1 marks early placode precursorcells similar to its expression during zebrafish development where itmarks the horseshoe shaped at the anterior pre-placodal region. thus theinventors discovered during the course of the present inventions thatIslet-1 is one of the first placode markers during differentiation ofhuman pluripotent stem cells. The modified N-SB protocol induced highlyenriched expression for Islet-1 at day 5, day 7, day 9 and day 11 whencompared to NSB conditions. In microarray data obtained by the inventorsduring the development of the present inventions, placode genes, GATA3,Dlx5, TFAP2a, and TFAP2c are enriched. The inventors also observedsignificant changes in Wnt pathway and BMP pathway components. As earlyas Day 5, there was a significant increase of the Wnt pathway inhibitorDKK-1 and this increase was sustained over the course of the protocol.The inventors also observed significant down-regulation of several Wntreceptors in response to removal of Noggin. Induction of BMP antagonistssuch as gremlin-1 and BAMBI that are activated in response to BMPsignaling confirm that noggin withdrawal causes changes in endogenousBMP signaling during hESC differentiation with a corresponding increaseof downstream genes.

Furthermore, The inventors identified a number of additional genes thatwere differentially expressed during anterior placode specificationincluding Shisa2, Ovol2 and Foxc1, Differential expression for these andadditional genes was verified by qRT-PCR. Ovol2 is a known marker ofsurface ectoderm and placodal fates in various model organisms. In mice,the Ovol2 knockout is lethal and Ovol2−/− mouse embryos do not developplacode-derived tissues such as optic cup, and otic vesicle.

A gene cluster analysis showed when genes were expressed the highest;time of maximum (TOM) and the lowest; time of minimum expression (TIM).GO ontology terms were mapped into this analysis and were able toidentify precise developmental windows during placode precursor cellspecification. These data additionally confirmed the identity of hESCderived placodal tissue and revealed markers and pathways involved inplacode versus anterior neural plate (AN) specification.

Clustering of differentially expressed transcripts (FIG. 12E) revealedcorrect matching of all replicate samples. These data also revealed aclose temporal matching of samples independent of treatment whilepinpointing to a subset of genes that distinguish neuroectodermal fromplacodal precursors (boxed area in FIG. 12E). Principle componentanalysis confirmed high temporal correlation of samples with increasingdivergence between neuroectodermal and placodal precursor cells at laterdifferentiation stages (FIG. 12F).

Placode Progenitors Give Rise to Sensory Neurons

Isolation of Six1+ placodal precursors followed by culture under serumfree conditions revealed efficient differentiation into neurons thatretain Six1 expression (FIG. 13A-C). The sensory neuron identity ofthese cells was confirmed by the expression of Brn3A, Isl-1 measured atday 20 of differentiation (FIG. 13D, E). Longer-term differentiationstudies (day 40) resulted in cells with strong expression of theperipheral neuron marker Peripherin (FIG. 13F) and reduced expression ofTuj1 compatible with in vitro maturation of sensory neuron progeny. Invitro developmental progression from placode precursor identity tomature sensory neuron fates is illustrated schematically in FIG. 13G.

Results showed that simple modifications in the N-SB protocol induce aswitch in differentiation from neuroectoderm to placodal precursors.Using timed-noggin withdrawal The inventors obtained a yield of 71% oftotal cells expressing Six1. The isolation of pure placodal precursorsrequires markers that prospectively identify placode fate. Theidentification of prospective markers is also critical to reliablydistinguish placodal cells from other alternative lineages such as CNSprecursor, neural crest lineages and non-neural ectoderm. Of particularinterest is the separation of placode derived from neural crest derivedprecursors to reliably separate neural crest from placode derivedneuronal populations. The inventors have previously demonstrated thatisolation of p75+/HNK1+ cells during neural early neural differentiationmarks a population of cells fated towards neural crest identity (Lee etal., Nature Biotechnology, 25(12):1468-75 (2007), herein incorporated byreference). Here The inventors tested the relationship of those markerswithin the placodal lineages. Interestingly, The inventors observed thatNGFR efficiently marks placodal cultures in our modified N-SB protocol(FIG. 14A) separated by populations of precursors expressing Forse1, amarker previously associated with anterior neuroectodermal fates(Elkabetz, G&D, 2008, herein incorporated by reference). Double sortingfor p75 and human natural killer-1 (HNK1) epitope (also known as CD57)expression revealed that p75-single positive cells, negative for HNK1,are dramatically enriched in Six1 expression based on qRT-PCR analysis(FIG. 14B, C). The placodal identity of the cells is further supportedby the increase in the number of p75+/HNK1− cells in theplacode-inducing modified N-SB protocol as compared to theneuroectoderm-inducing classic N-SB protocol (FIG. 14D).

Example II IPS Cell Generation.

The cDNAs encoding hOct4, hSox2, hKlf4 and c-myc (purchased from OpenBiosystems) were subcloned into self-inactivating lentiviral vectorsdriven by the human phosphoglycerate kinase (PGK) promoter. Lentiviralvector supernatants were produced by triple co-transfection of theplasmid DNA encoding the vector, pCMVΔR8.91 and pUCMD.G into 293T cells.Human fetal lung fibroblasts (MRC-5) purchased from ATCC (CCL-171) wereseeded at 1.5×10⁴ cells/cm² in Eagle's Minimum Essential Mediumsupplemented with 10% fetal bovine serum (FBS). The following day thefibroblasts were transduced with equal amounts of supernatants of thefour lentiviral vectors in the presence of 4 ug/ml polybrene for ˜16hours. Six days after transduction, fibroblasts were harvested bytrypsinization and plated at 2×10⁴ cells per 60 mm dish on a feederlayer of mytomycin C-treated mouse embryonic fibroblasts (CF-1). Thenext day, the medium was switched to hESC medium. The hiPS lines wereconfirmed positive for Tra-1-81, Tra-1-60, SSEA-4 and Nanog byImmunoflouresence and flow cytometry. In both hips clones the 4vector-encoded transgenes were found to be silenced.

Materials and Methods:

Cells and Culture Conditions (dual SMAD and floor plate). hESCs (WA-09;passages 35-45) were cultured on mouse embryonic fibroblasts plated at12-15,000 cells/cm2 (MEFs, Global Stem). A medium of DMEM/F12, 20%knockout serum replacement (GIBCO), 0.1 mM b-mercaptoethanol, 6 ng/mLFGF-2 was changed daily. Cells were passaged using 6 U/mL of dispase inhESCs media, washed and re-plated at a dilution of 1:5 to 1:10.

Neural Induction (Dual SMAD).

hESC cultures were disaggregated using accutase for 20 minutes, washedusing hESC media and pre-plated on gelatin for 1 hour at 37° C. in thepresence of ROCK inhibitor to remove MEFs. The nonadherent hESC werewashed and plated on matrigel at a density of 10,000-25,000 cells/cm² onmatrigel (BD) coated dishes in MEF conditioned hESC media (CM) spikedwith 10 ng/mL of FGF-2 and ROCK-inhibitor. Ideal cell density was foundto be 18,000 cells/cm². The ROCK inhibitor was withdrawn, and hESC wereallowed to expand in CM for 3 days or until they were nearly confluent.The initial differentiation media conditions included knock out serumreplacement (KSR) media with 10 nM TGF-beta inhibitor (SB431542, Tocris)and 500 ng/mL of Noggin (R&D). Upon day 5 of differentiation, the TGF-binhibitor was withdrawn and increasing amounts of N2 media (25%, 50%,75%) was added to the KSR media every two days while maintaining 500ng/mL of Noggin. For MS5 induction, established methods previouslyreported were used.¹⁸

Quantitative Real-Time (Dual SMAD).

Total RNA was extracted using an RNeasy kit (Qiagen). For each sample, 1ug of total RNA was treated for DNA contamination and reversetranscribed using the Quantitect RT kit (Qiagen). Amplified material wasdetected using Quantitect SYBR green probes and PCR kit (Qiagen) on aMastercycler RealPlex2 (Eppendorf). Results were normalized to a HPRTcontrol and are from 4-6 technical replicates of 2-3 independentbiological samples at each data point.

Neuronal Patterning and Differentiation (Dual SMAD).

Dopaminergic patterning was initiated using BDNF, ascorbic acid, sonichedgehog, and FGF8 in N2 media as previously reported¹⁸, and maturationwas performed in the presence of BDNF, ascorbic acid, GDNF, TGFb-1, andcyclic-AMP. Motor neuron acid in N2 media as previously reported.¹⁶

Microscopy, Antibodies, and Flow Cytometry (Dual SMAD).

Tissue was fixed using 4% paraformaldehyde for 20 minutes, washed withPBS, permeablized using 0.5% Triton X in PBS, and blocked using 1% BSAin PBS. Primary antibodies used for microscopy included PAX6 (Covance),Oct4 (Biovision), AP2 (Novus Biologicals), GBX2 (Sigma), HNK1 (Sigma),HOXB4 (Developmental Studies Hybridoma Bank (DSHB)), Nestin (R&D),NKX6.1 (DSHB), OTX2 (gift), p75 (Advanced Target Systems.), PAX7 (DSHB),PLZF (Calbiochem), TUJ1 (Covance), ZO1 (Zymed), BF1 (FOXG1, gift EssengLai), TH (Sigma), HB9 (DSHB), ISL1 (DSHB). CD105-PE (eBioscience) wasused for excluding MS5 stromal cells for flow cytometery on a FACScan(BD).

Floor Plate: Neural Induction.

For MS5 induction, established methods previously reported were used(Perrier et al., 2004). Feeder free neural induction was carried out aspreviously described (Chambers et al., 2009). Briefly, hESCs cultureswere disaggregated using accutase for 20 minutes, washed using hESCsmedia and pre-plated on gelatin for 1 hour at 37° C. in the presence ofROCK inhibitor to remove MEFs. The nonadherent hESCs were washed andplated on matrigel at a density of 20,000 cells/cm2 on matrigel (BD)coated dishes in MEF conditioned hESCs media (CM) spiked with 10 ng/mLof FGF-2 and ROCK-inhibitor. The ROCK inhibitor was withdrawn, and hESCswere allowed to expand in CM for 3 days or until they were nearlyconfluent. The initial differentiation media conditions included knockout serum replacement (KSR) media with 10 nM TGF-b inhibitor (SB431542,Tocris) and 500 ng/mL of Noggin (R&D). Upon day 5 of differentiation,increasing amounts of N2 media (25%, 50%, 75%) was added to the KSRmedia every two days while maintaining 500 ng/mL of Noggin and TGF-binhibitor. For FP induction, Sonic C25II was added at 200 ng/ml. In someexperiments, DKK-1 (R&D 100 ng/ml) FGF8 (R&D 50 ng/ml), Wnt-1 (Peprotech50 ng/ml) and Retinoic Acid (R&D 1 uM) were added.

Quantitative Real-Time PCR.

Total RNA was extracted using an RNeasy kit (Qiagen). For each sample, 1ug of total RNA was treated for DNA contamination and reversetranscribed using the Superscript III (Invitrogen). Amplified materialwas detected using Taqman probes and PCR mix (ABI) on a MastercyclerRealPlex2 (Eppendorf). All results were normalized to a HPRT control andare from 3 technical replicates of 3 independent biological samples ateach data point.

Microarray Analysis.

Total RNA was isolated at Days 2, 3, 5, 7, and 11 of differentiationfrom both control (NSB) and FP (NSB+Shh C25II) using Trizol(Invitrogen). Three biological replicates per time point were used. Allsamples were processed by the MSKCC Genomics Core Facility andhybridized on Illumina human 6 oligonucleotide arrays. Normalization andmodel-based expression measurements were performed with using theIllumina analysis package (LUMI) available through open-sourceBioconductor project (www.bioconductor.org) with in the statisticalprogramming language R (http://cran.r-project.org/). A pairwisecomparison between NSB and

NSB+Sonic was performed using the Linear Models for Microarray Datapackage (LIMMA) available through Bioconductor. Genes found to have anadjusted p-value <0.05 and a fold change greater than 2 were consideredsignificant. Expression differences are reported as the log 2 of thefold change. Gene Ontology enrichment was determined by entering genelists into the Database for Annotation, Visualization, and IntegratedDiscovery (DAVID; http://www.david.niaid.nih.gov) (Huang et al., 2009and Dennis et al., 2003). Timing of maximal and minimal expression wascalculated as previously reported (Venezia et al., 2004).

Briefly, a regression line was fit to both the NSB+Sonic C25II and NSBconditions. From these trend lines, genes were categorized based on atwhich time point its maximal and minimal expression occurred.

Microscopy, Antibodies, and Flow Cytometery.

Tissue was fixed using 4% paraformaldehyde and Picric acid for 15minutes, washed with PBS, permeablized using 0.3% Triton X in PBS, andblocked using 10% Donkey Serum. Primary antibodies used for microscopyincluded PAX6 (Covance), TUE (Covance), ZO1 (Zymed), BF1 (FOXG1, giftE.Lai), TH (Pelfreez), NKX6.1 (DSHB) and FOXA2 (SantaCruz).

Vector Design and Lentiviral Production.

A third generation lentiviral vector (Lois et al., 2002) was modified toexpress a BF1 ORF from the Ub-C promoter (Fasano et al., 2009) and a BF1shRNA from the H1 promoter as described (Fasano et al., 2007; Ivanova etal., 2006). Foxg1 shRNA constructs were used as previously described(Shen et al., 2006). The shRNA expressing lentiviral plasmid wasco-transfected with plasmids pVSV-G and pCMVd8.9 into 293FT cells. Viralcontaining media were collected, filtered, and concentrated byultracentrifugation. Viral titers were measured by serial dilution onNIH 3T3 cells followed by flow cytometric analysis after 72 hours.

Generation of BF1 shRNA and Over-Expressing Human ES Lines.

hESCs (WA-09; passages 35) were dissociated and plated on Matrigel withthe ROCK inhibitor as singles cells. 24 hrs post plating the ES cellswere transduced with either control (empty vector), BF1 shRNA, or BF-1ORF containing vectors. 1 week later, GFP expressing colonies weremanually picked and plated on MEFs. Cells were then expanded, tested formycoplasma, and a normal karyotype.

Dissection of Primary Explants.

E8.5, TP Taconic Swiss Webster females were dissected and embryos wereremoved. Neuroectodermal tissues were dissected and left as chunksplated on top of FP cells. For neurite growth assay E12.5 Sprague-Dawleyrat cerebellar plate tissue was dissected and plated on top of hESCderived FP cells or control neuroectodermal cells (NSB protocol).Outgrowth from rat explants tissue was analyzed at day 3 of co-culture.

Conditioned Media and ELISA.

hESCs were differentiated to neural or FP cells, Shh was removed a day6, and the media was harvested at both day 9 and day 11 of cultures.Using a human Netrin-1 ELISA kit (Axxora) according to the manufacturesprotocol, Netrin-1 protein levels were detected. For co-cultureexperiments, the media was filtered and added to cultures straight or a1:2 dilution in fresh media.

Statistical Analysis.

Results shown are mean+s.e.m. Asterisks and pound signs identifyexperimental groups that were significantly different from controlgroups by a t-test, one way ANOVA, or two way ANOVA with a Bonferronicorrection for multiple comparisons (p-value, 0.05), where applicable.

Example III Early High-Dose SHH Exposure Induces FOXA2 and RepressesBF1.

hESC derived neural cells at the rosette stage were differentiated intoboth CNS and PNS progeny and patterned towards multiple cells fatesalong the A/P and D/V axis (Elkabetz et al., 2008). These resultsdemonstrated that rosette stage cells were highly plastic and responsiveto patterning cues including SHH. Specification of progenitor cells intoFP tissue and cells during mouse development was thought to depend onSHH signaling within early neural lineages. The inventors tested whetherrosette-stage neural cells were competent to undergo FP specification inresponse to SHH. High concentrations of SHH were needed to induce FPduring mouse development (Roelink et al., 1994; Ericson et al., 1996).Recombinant N-terminal SHH has a limited activity range due to the lackof posttranslational modifications required for full SHH action.Recently, a modified version of recombinant SHH became available whereSHH was tethered to two Isoleucines (Sonic C25II, R&D Systems),mimicking more closely the potency of mammalian SHH protein. In mostfunctional assays C25II was ˜10 times more potent than non-modifiedN-terminal SHH.

However, dose-response studies done during the development of thepresent inventions with both conventional SHH and SHH-C25II onestablished rosette-stage neural cells did not yield cells expressing FPmarkers such as FOXA2 (FP marker) under any of the conditions tested.The majority of cells retained rosette cytoarchitecture and staining forthe AN marker BF1 as described previously (FIG. 1A) (Elkabetz et al.,2008). These results were a surprise, i.e. exposure to high SHH was notsufficient to convert established rosette-stage cells into FP.

Based on the hypothesis that FP specification in the mouse occurs atearly developmental stages, at the time of or prior to neural induction,the inventors repeated SHH induction studies at Day 9, the time ofrosette specification, using classic stromal-feeder mediated neuralinduction protocols (Elkabetz et al., 2008). Under this paradigm theinventors noticed a drastic change in cell morphology restricted to thecells treated with SHH-C25II (FIG. 1B). Cells exhibited a flatmorphology devoid of rosette structures. Furthermore, the inventors'observed a robust upregulation of FOXA2+ and a concomitant decrease inBF1+ cells (FIG. 1C) and the decrease in the total number of ZO1+rosettes (FIG. 1D). In addition to decreased expression of BF1 theinventors also observed decreased expression of PAX6, another makerexpressed in the AN (FIG. 1E). Dose-response studies demonstrated thatinduction of FOXA2+ cells and the concomitant decrease in BF1 and PAX6expression were achieved at concentrations of 125-500 ng/ml of SHH C25II(FIG. 1F and not shown). No efficient induction of FOXA2+ cells wasobserved with any of the concentrations tested using non-modifiedN-terminal SHH. The inventors performed dose response studies tounderstand how FP marker induction compared to that of NKX6.1expression; a gene known to respond to lower concentrations of SHH. Atlow concentrations of Sonic C25II there is no expression of FP markersFOXA2 and Netrin-1 but a robust increase in NKX6.1 expression. At higherconcentrations FP markers rapidly rise, while NKX6.1 expression tapersoff (FIG. 1G). These data demonstrated that early exposure to highlevels of SHH decreases anterior AN markers and induces the FP markerFOXA2.

Example IV The Competency for FP Induction is Restricted to a NarrowWindow of Differentiation.

While a robust (strong observed signal, such as staining) upregulationof FOXA2 was induced with the initial procedures, merely around 30% ofthe cells were positive after about 21 days of culture using classicstromal-feeder mediated neural induction. Therefore the inventors testedseveral types of culture compositions and methods for increasing thetotal number of cultured cells expressing FOXA2.

Recently the inventors developed and described a rapid and definedneural induction paradigm yielding significantly higher numbers ofneural cells based on inhibiting SMAD signaling via exposure to nogginand SB431542 (NSB protocol; (Chambers et al., 2009)). Using thisprotocol, in combination with compositions and methods of the presentinventions, the inventors aimed to optimize FP differentiation by addingSonic C25II at different time points during neural induction andassaying for FOXA2 expression. Differentiation was initiated upon NSBexposure, and Sonic C25II was added at Day 1, Day 3, Day 5, or Day 7(FIG. 2A). The most efficient FOXA2 induction was observed in culturestreated with SHH starting at day 1 of differentiation with FOXA2+ cellsrepresenting about 65% of total cells (FIGS. 2B and 2C). Extended SHHtreatment beyond Day 11 of differentiation did not increase FOXA2 yield(FIG. 2D). These data demonstrated that an early high SHH signal isneeded to establish FP identity and suggest a critical window ofcompetency for FP specification. Furthermore, the differentiationconditions establish a robust platform for inducing human FOXA2+ cellsin vitro.

FOXA2 is a key marker of FP development. However, FOXA2 is also highlyexpressed in the endoderm. To further characterize the hESC derivedputative FP tissue, the inventors performed qRT-PCR analyses forcandidate markers at Day 11. Using the NSB protocol as a control, theinventors confirmed a dramatic increase in the expression of FOXA2 andother FP markers including SHH, F-Spondin, and Netrin-1 (FIG. 7). Theinventors further characterized the nature of FOXA2+ putative FP cellsusing a panel of neural precursor, glial, neuronal and non-neuralmarkers (FIG. 7). FOXA2+ cells co-labelled with only a limited subset ofthese markers including Nestin (86%) and SOX2 (17%). To distinguishFOXA2 expression in hESC derived FP versus endoderm tissue, theinventors differentiated hESCs to endoderm (D'Amour et al., 2005). Asexpected, under both FP and endoderm differentiation conditions, theinventors observed an increase in FOXA2 expression compared with NSBtreated control cells. However, induction of the endoderm marker SOX17was limited to the endoderm condition and no SOX17 was present in hESCderived FP cells (FIG. 7). The inventors also did not observe expressionof other endodermal markers such as AFP and Albumin expression in hESCderived FP cells. These data demonstrated that hESC derived FOXA2+ cellsin the NSB+SHH protocol express FP and early neural precursor markersand lack expression of endodermal markers.

Example V

hESCs Derived FP Cells are Functional.

The FP has important functional roles during development in neuralpatterning and axonal path finding (Jessell, 2000). To assess thefunctional properties of hESCs derived FP conditioned media was isolatedat days 9 and 11 and tested for expression of Netrin-1 in the mediumusing ELISA (FIG. 3A). Under normal NSB conditions, Netrin-1 isdetectable at Day 9 and decreases at Day 11 while in the NSB+SHHcondition, there is a 3.5 fold increase in Netrin-1 levels, increasingat Day 11 (FIG. 3B). SHH is a critical patterning factor secreted by FPcells and specifying ventral cell types in a dose-dependent manner. Totest if hESCs derived FP secretes factors that can specify ventralprecursor domains, conditioned media (CM) was isolated at Days 9 and 11of the differentiation. At Day 6, exogenous SHH was removed and cultureswere washed to eliminate any exogenously added SHH from the medium.Naïve neural progenitor cells were isolated at Day 11 of the control(NSB) protocol and cultured with either NSB CM or FP CM. After Day 5 ofculture in the presence of CM, the inventors probed for the expressionof ventral precursor markers and expression of the SHH responsive geneGLI2. The inventors found that compared with CM obtained from NSBcontrol cultures, CM from hESC derived FP tissue efficiently inducedexpression of ventral genes including NKX2.1 and NKX6.1 (FIG. 3C).Increase in the expression of ventral markers was confirmed at the levelof protein (FIG. 3C′).

To see if this result was SHH-mediated, the inventors demonstratedincreased expression of GLI2 upon exposure to CM from hESC derived FP.When this experiment was repeated in the presence of the SHH antagonistcyclopamine, all three genes, NKX2.1, NKX6.1, and GLI2 weresignificantly reduced (FIG. 3C) demonstrating dependence of patterningresponse on SHH signaling.

Classical studies demonstrated that FP explants can induce an ectopic FPin early neuroectodermal tissue (Placzek et al., 1993). To test if thehESCs derived FP is capable of inducing FP markers in primary mouseexplants, neuroectodermal tissue was isolated from an E8.5 mouse embryoand placed it in direct contact with hESC derived FP cells. After 3 daysof co-culture explants were identified based on expression of the mousespecific M6 marker, rinsed and mounted on slides to be stained forFOXA2. As a control condition, mouse explants were co-cultured with hESCderived neural tissue using the NSB protocol. While co-culture withcontrol hESC derived neural tissue did not yield FOXA2+ cells, explantsco-cultured with hESC derived FP cells showed robust induction of FOXA2+cells, particularly at the periphery of the explant (FIGS. 4D and 4E).Neurite growth promoting effects of hESC derived FP cells was observedin primary rat E12.5 rat cerebellar plate explants (FIG. 8), an assayused previously to demonstrate axonal growth promoting effects ofprimary rodent FP tissue (Shirasaki et al., 1995). These experimentsdemonstrated that hESCs derived FP can mimic the functional propertiesof primary FP tissue as an organizer by secreting Netrin-1 and SHHcapable of ventralizing naïve hESC derived and primary mouse neuralprecursor cells.

Example VI

Temporal Transcriptome Analysis Reveals that FP Specification Occurs atthe Expense of AN.

To gain further insight into the factors critical for human FPspecification, high resolution temporal gene expression profiles ofcandidate markers were performed at 6 time points during the 11 dayprotocol. FOXA2 expression was observed as steady increase in transcriptlevels starting at day 3 of differentiation (compared to NSB) consistentwith immunostaining data (FIG. 4A). Interestingly, other FP markers;SHH, Netrin-1, and F-Spondin followed a different expression pattern(FIG. 4B-4D). All three markers showed a more delayed induction with adramatic increase in expression (compared to NSB condition) at day 7 ofdifferentiation.

PTCH1 expression is used commonly as a transcriptional readout of SHHactivity. Dramatic increase in PTCH1 expression was observed as early asday 3 of differentiation with levels further increasing by a factor of 3over the next 2 days (FIG. 4E). It has been shown previously that theSHH downstream effector GLI2 is essential for FP induction but decreasesat later stages of FP development (Matise et al., 1998) and that GLI2can directly activate FOXA2 expression (Jeong and Epstein, 2003). Anearly increase in both GLI2 and FOXA2 expression (FIGS. 4A and 4F) wasobserved followed by a decrease in GLI2 at Day 11 consistent with a roleof GLI2 specifically during FP induction. A similar trend albeit at muchlower induction levels is observed for GLI1 (FIG. 4G).

SOX1 is an early neural marker and is not expressed in the medial FP(Charrier et al., 2002). Consistent with a rapid neural induction, SOX1was rapidly up regulated in NSB conditions and continued to increasewith time. Upon addition of SHH a much smaller increase in SOX1 levelsis observed at day 3 compared with control NSB conditions (FIG. 4H). NSBconditions yield neural cells with a AN bias expressing BF1 at highlevels (Chambers et al., 2009). However, when SHH is added to theculture, there is a drastic reduction in PAX6 and BF1 at day 7 (FIGS. 4Iand 4J). Induction of the endoderm marker SOX17 and mesoderm markerBrachury was not observed (FIGS. 4K and 4L) suggesting that FPinduction, similar to AN induction using the NSB protocol, occurswithout contribution of an obvious mesodermal or endodermalintermediate. These data demonstrated appropriate marker expression inhESC derived FP, initiated by GLI2 and FOXA2 expression and followed byexpression of functional FP markers such as Netrin-1, SHH, andF-Spondin. The drop in PAX6 and BF1 expression at the time of FPspecification suggests that induction of FP occurs at the expense of AN.

Example VII

Global Transcriptome Analysis During hESC Derived FP Specification.

Temporal profiles of global gene expression at 5 time points duringdifferentiation was established during the development of the presentinventions (Day 1, 3, 5, 7 and 11) in control NSB cultures (yielding AN)and in Sonic C25II treated cultures (yielding FP; see FIG. 2E). Prior tomicroarray analysis the quality of each sample was verified forexpression of a panel of FP markers (FIG. 9). Global gene expressionstudies were carried out in three independent samples for each timepoint and culture condition. Data were converted into log 2 ratioscomparing levels of gene expression in FP versus NSB protocol duringdifferentiation (FIGS. 4I-4Q). Raw data are available in GEO database(http://www.ncbi.nlm.nih.gov/geo/) accession number: GSEXXX (numberavailable at time of publication).

The time course data were subjected to gene ontology (GO) enrichmentanalysis using DAVID (http://david.abcc.ncifcrf.gov/; Dennis et al.,2003) as unbiased assessment of the FP transcriptional profile. Amongthe transcripts highly enriched in SHH treated versus NSB controlcultures at day 7 and 11 of differentiation were genes associated withthe Wnt and hedgehog pathways, axon guidance, and secreted proteins(FIGS. 4L and 4M). Enrichment for patterning and axonal guidance factorsfurther confirm FP identity of SHH treated cultures. Further,SHH-mediated suppression of AN was demonstrated when transcripts thatincluded genes involved in forebrain development showed a larger amountof downregulation in the FP culture methods for producing floor platecells than when compared to cells cultured with the NSB protocol (FIG.4L, M).

Pairwise comparisons at for each differentiation stage was done to gaininsight into specific genes differentially expressed during FPspecification. While the majority of genes significantly regulated atday 3 and day 5 of differentiation (as compared to day 1) were shared inNSB and FP protocol, a subset of transcripts was differentiallyregulated (FIGS. 4N-4Q). In particular, an increase in Patched-1(PTCH1), a component and known transcriptional downstream target of theSHH signaling was noticed. In this protocol, PTCH1 is highly enriched atall time points, except D11 where it starts to decrease (FIG. 4N-4Q).

The inventors observed significant changes in the Wnt pathwaycomponents. As early as Day 5 there was a significant decrease of theWnt pathway inhibitor DKK-1 and this decrease was sustained over thecourse of the protocol (FIG. 4N-4Q and FIG. 7). Significant upregulationof several Frizzled genes that have been previously shown to be involvedin midline axon guidance during mouse development (Lyuksyutova et al.,2003) in the midline (FIGS. 4P and 4Q) was also observed. Additionally,a number of additional genes were identified that were differentiallyexpressed during FP specification including SIX6, CAPN6, IGFBP3 andFIBLN1 (FIGS. 4N-4Q). Differential expression for these and additionalgenes was verified by qRT-PCR (FIG. 9). While systematic in situhybridization screens in mouse and human embryonic tissue will berequired to validate putative human FP markers, based on the literatureand MGI (Mouse gene expression database), many of the genes identifiedhave compatible expression patterns in the anterior midline and floorplate tissue such as HESX1 (Zoltewicz et al., 1999) or RBP1(CRBP1—Hunter et al., 1991) respectively.

Example VIII

A gene cluster analysis was also done that showed when genes areexpressed the highest; time of maximum (TOM) and the lowest; time ofminimum (TIM) expression. GO ontology terms were mapped during thisanalysis and were able to identify precise developmental windows duringthe FP specification process. These data further confirmed the identityof hESC derived FP tissue and provides insight into genes differentiallyexpressed during FP versus neuroectodermal fate specification.

Example IX Suppression of DKK-1 Blocks AN Commitment and Enhances FPGeneration.

The inventors observation that FP commitment occurs at the expense of ANwas strengthened by the global gene expression profiles obtained hereinthat revealed a rapid down regulation of the Wnt signaling inhibitorDKK-1. DKK-1 was initially identified as a factor expressed in thexenopus head organizer that was necessary and sufficient to induce headdevelopment (Glinka et al., 1998). DKK-1-mediated inhibition of Wntsignaling during mouse development is essential for anterior braindevelopment (Mukhopadhyay et al., 2001), and FOXA2 knockout embryos showincreased expression of DKK-1 in the ectoderm at E7.5 (Kimura-Yoshida etal., 2006). During NSB induction it was observed that DKK-1 transcriptlevels rise sharply at day 5 from 200 to 5000 fold and then drop backdown consistent with the role of DKK-1 as an AN inducer. ELISA assayswere done to measure DKK-1 protein levels in the medium and found levelsas high as 12 ng/ml (FIGS. 5A, B). A drastic reduction of DKK-1 at bothmRNA and protein levels was observed as early as 2 days post Sonic C25IItreatment (FIG. 5B,C). The decrease in DKK-1 expression was sustainedand accompanied by decreases in AN markers including PAX6, BF1, OTX1,OTX2, and EMX1 (FIG. 4).

To test whether DKK-1 is functionally involved during hESCdifferentiation in FP specification, the inventors added recombinantDKK-1 in combination with Sonic C25II and assessed FP marker expression.While treatment with Sonic C25II alone resulted in a decrease of the ANmarker BF1 and an upregulation of FOXA2 (FIG. 5D-F), the addition ofDKK-1 caused a decrease in FOXA2 message and protein and a more rapidrise in BF1 transcript (FIG. 5D-F). Conversely, addition of DKK-1antibody to cells in the NSB protocol caused a significant delay anddecrease in the levels of BF1 expression. These data indicated thatendogenous DKK-1 levels are critical for AN specification. Next, hESCswere differentiated in the presence of both Sonic C25II and DKK-1neutralizing antibody. Under these conditions, early transient inductionof BF1 transcript at day 5 is suppressed and accompanied by an increasein FOXA2 levels (FIGS. 5E and 5G).

The data obtained during the development of the present inventionsrevealed a critical window for FP specification during neural induction.With the observation that DKK-1 expression can inhibit FOXA2 expression,the following test was designed to demonstrate whether the addition ofDKK-1 blocking antibody extends the window of competency for SHHmediated FP induction. DKKK-1 antibody was added at Day 1, Day 5, andDay 9 of differentiation. Exposure to SHH was initiated at the same timepoints and the expression of FOXA2 was assayed following 9 days of SHHexposure. When Dkk-1 was added along with SHH at Day 1 an increase inFOXA2+ cells was observed. However, when added at Day 5 or Day 9, DKKK-1antibody FOXA2+ cells were not observed (FIGS. 5H and 5I). These dataindicate that high, early endogenous levels of DKK-1 in the NSB protocolinitiated AN commitment and suppressed FP competency. Early treatmentwith SHH repressed DKK-1 mediated AN specification and enableddifferentiation towards FP lineage. However, inhibition of DKK-1 at day5 of later stages did not extend the temporal window for FP induction.

Example X Bf1 Expression Represses Fp Commitment.

The inventors discovered that SHH addition to stem cell cultures causedFP differentiation at the expense of AN, mediated at least in part,through inhibition of DKK-1. DKK-1 was shown to specify BF1+neurectoderm BF1 (Mukhopadhyay et al., 2001), and BF1 is expressed inmost neural cells upon NSB induction (FIGS. 4 and 5). To test whetherexpression of the forkhead factor BF1 directly represses FP competencyduring neural induction, hESCs were transduced with a BF1 shRNAconstruct (Fasano et al., 2009; Shen et al., 2006) and clonal lines werederived. BF1 is not highly expressed in hESCs and there was nodifference in cell morphology or colony size (FIG. 10)).

However, upon neural differentiation of hESCs there was a decrease inBF1 protein expression (FIGS. 5J″ and 5K″) and an 80% decrease in BF1transcript (FIG. 10). While BF1 loss of function has been associatedwith deficits in proliferation and cell cycle progression at the neuralprecursor stage, BF1 knockdown lines at the hESC stage showed cell cyclekinetics comparable to control vector transduced lines (FIG. 10). BF1knock-down and control hESCs were then differentiated to FP andsubjected to qRT-PCR analysis for a panel of FP markers. After 11 daysof differentiation, there was as significant increase in expression ofall FP markers in the BF1 shRNA condition (FIG. 5L). Furthermore,immunocytochemical analyses revealed a significant increase in thenumber of FOXA2+ cells, representing greater than 90% of total cells inthe BF1 knockdown hESC line (FIG. 5M).

hESC lines were generated by overexpression of BF1 using a previousdescribed vector (Fasano et al., 2009). These transgenic cells were thencultured under conditions that induced differentiation towards FPlineage. At Day 11, compared to a control GFP expressing clones, therewas a reduction of FOXA2+ cells and a decrease in FP marker expression(FIG. 10). These data demonstrated that BF1 expression inhibited thederivation of hESC derived FP.

Example XI

The A/P Axis of the FP were Altered by Caudalizing Agents.

While certain characteristics are shared among all FP cells, such asFOXA2 and Netrin-1 expression, differences have been reported betweendifferent regions of the floor plate along the A/P axis (Placzek andBriscoe, 2005). In particular, recent studies have shown that themidbrain FP expresses markers such as CORIN (Ono et al., 2007) and NOV(Placzek and Briscoe, 2005).

Additionally, the midbrain FP was shown to be neurogenic giving rise tomidbrain DA neurons and expressing markers of DA progenitors such asLMX1B and NGN2 (Joksimovic et al., 2009). In contrast both the hindbrainand spinal cord FP appear to be non-neurogenic. To better understand theA/P identity of the FP cells generated from hESCs qRT-PCR analysis forthe midbrain FP markers CORIN and NOV was done, as well as analysis ofDA progenitor markers LMX1B and EN1. However expression of these markerswas not detected. Next gene expression data sets were used to identifydifferentially regulated transcripts markers that could shed light ontothe positional identity of the FP cells.

Elegant studies in the mouse showed that specific enhancer elementsdirect Shh expression in different regions along the A/P axis of the FP(Jeong et al., 2005). SIX6 were dramatically increased during FPinduction compared with NSB control conditions (About 50,000-foldincrease in mRNA levels at Day 5 of differentiation; FIG. 9). SIX6 hasbeen shown to bind to the SHH gene at an enhancer region known as SBE2that directs SHH expression to the most anterior aspect of the ventralbrain (Jeong et al., 2008). The inventors contemplated the use of SIX6as a putative marker of anterior FP identity. Thus SIX6 status of thehESC derived FP was used to mark respecification in response to knowncaudalizing agents such as FGF8, Wnt-1, and Retinoic Acid. Each of thesecaudalizing factors was added in combination with SHH and the resultingtissue was assessed for expression of the FP markers FOXA2 and NETRIN-1,the AN marker BF1, and putative anterior FP marker SIX6. FP generationwas found not compromised in the presence of caudalizing factors. Infact, the addition of Wnt-1 or RA significantly potentiated FPproduction based on FOXA2 and Netrin-1 expression. (FIGS. 6A and 6B).Enhanced expression of FP markers in the RA and Wnt-1 group wascorrelated with a dramatic reduction in BF1 expression furthersupporting the notion that AN commitment counteracts FP induction (FIG.6B). Strikingly, in all conditions, there was a significant reduction inSIX6 expression, with the Wnt-1 and RA conditions being the mosteffective at suppressing anterior FP identity.

Example XII

This example shows exemplary experiments designed to determine whetherany of the methods (conditions) used herein would lead to anupregulation of midbrain FP and DA progenitor markers. The inventorsdiscovered that different factors had varied effects on markerexpression (FIG. 6B). In particular, exposure to Wnt-1 resulted in asignificant increase in the midbrain FP markers CORIN and NOV, as wellas increases in the DA progenitor markers LMX1B, EN1, and NGN2. Previousstudies showed that Wnt signaling was critical in the neurogenicresponse of the midbrain FP (Joksimovic et al., 2008).

As mentioned above, studies had identified different enhancers thatdirected SHH expression to different A/P region along ventral axis(Jeong et al., 2008). To further demonstrate that the addition ofcaudilizing factors re-specifies A/P identity of the resulting FPtissue, hESCs derived FP were generated in the presence or absence ofWnt-1 or FGF8, and transfected the resulting tissue with two SHHenhancer constructs driving LacZ expression in different A/P domains ofthe FP. The SBE1 construct directs SHH expression to the midbrain regionof the floor plate while the SBE2 enhancer directs SHH expression to themost anterior region of the FP where Six6 has been shown to bind. In theabsence of caudalizing factors (SHH C25II alone), LacZ expression wasobserved herein following transfection with the SBE2 but not the SBE1enhancer supporting the hypothesis that hESC derived FP is anterior bydefault (FIG. 6C). In contrast SBE2 activity was abolished upontreatment with Wnt1 or FGF8 while SBE1 activity was induced under theseconditions. These data indicated that FGF8 or Wnt1 treatment induces ashift in FP identity towards a more caudal, midbrain-like identity (FIG.6C).

In conclusion, our data demonstrated that upon neural differentiationhESCs default towards an AN fate by upregulating DKK-1 and subsequentlyBF1, and that AN commitment actively represses FP competency in hESCprogeny. However, an early high level of SHH reduces DKK-1 levelsenabling FP induction at the expense of AN while loss-of-function ofDKK-1 or BF1 increases FP production. Human ESC derived FP is anteriorby default but were posteriorized in response to caudalizing factors.This is summarized in FIG. 6E.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in cellularbiology, neurobiology, cancer cell biology, molecular biology,biochemistry, chemistry, organic synthesis, or related fields areintended to be within the scope of the following claims.

We claim:
 1. A method for inducing differentiation in stem cells, comprising, a) providing: i) a cell culture comprising human pluripotent stem cells, ii) a first inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling, wherein said first inhibitor is selected from the group consisting of Noggin, a disulfide-linked homodimer of Noggin, Dorsomorphin, LDN-193189, and mixtures thereof, and iii) a second inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling, wherein said second inhibitor is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl] benzamide (SB431542), and b) contacting said human pluripotent stem cells with said first inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling and said second inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling, and c) inducing differentiation of said contacted pluripotent stem cells into a population of cultured neuroectodermal precursor cells.
 2. The method of claim 1, wherein said contacting is performed at least 72 hours after said providing said cell culture.
 3. The method of claim 1, wherein said cell culture comprises an N2 culture medium.
 4. The method of claim 1, wherein said method further comprises a step of removing said first inhibitor from said contacted pluripotent stem cells within 48 hours after contacting said contacted pluripotent stem cells with said first inhibitor wherein said population of cultured neuroectodermal precursor cells comprise placode precursor cells.
 5. The method of claim 4, wherein said placode precursor cells further differentiate into Brn3a+ progenitor cells under serum free conditions.
 6. The method of claim 1, wherein said population of neuroectodermal precursor cells further differentiate into cells selected from the group consisting of central nervous system (CNS) progenitor cells, patternable neuronal cells, dopamine positive neurons and motoneurons.
 7. The method of claim 1, further comprising a step of contacting said plated pluripotent stem cells with, a modified recombinant protein at least 99% identical to a mouse Sonic Hedgehog N-terminal fragment within 60 hours of said contacting of said first inhibitor and said second inhibitor, wherein said population of neuroectodermal precursor cells further differentiate into a posterior floor plate tissue or floor plate cells.
 8. The method of claim 1, wherein said neuroectodermal precursor cells are at least 10% up to 100% of said population of contacted pluripotent stem cells.
 9. A method for inducing differentiation in stem cells, comprising, a) incubating a cell culture comprising human pluripotent stem cells with a culture medium, b) contacting said human pluripotent stem cells with a first inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling, wherein said first inhibitor is selected from the group consisting of Noggin, a disulfide-linked homodimer of Noggin, Dorsomorphin, LDN-193189, and mixtures thereof and a second inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling, wherein said second inhibitor is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl] benzamide (SB431542) and c) inducing differentiation of said contacted pluripotent stem cells into a population of cultured neuroectodermal precursor cells.
 10. The method of claim 9, wherein said contacting is performed at least 72 hours after said incubating.
 11. The method of claim 9, wherein said cell culture comprises an N2 culture medium.
 12. The method of claim 9, wherein said method further comprises a step of removing said first inhibitor from said contacted pluripotent stem cells within 48 hours after contacting said contacted pluripotent stem cells with said first inhibitor wherein said population of cultured neuroectodermal precursor cells comprise placode precursor cells.
 13. The method of claim 12, wherein said placode precursor cells further differentiate into Brn3a+ progenitor cells under serum free conditions.
 14. The method of claim 9, wherein said population of neuroectodermal precursor cells further differentiate into cells selected from the group consisting of central nervous system (CNS) progenitor cells, patternable neuronal cells, dopamine positive neurons and motoneurons.
 15. The method of claim 9, further comprising a step of contacting said plated pluripotent stem cells with, a modified recombinant protein at least 99% identical to a mouse Sonic Hedgehog N-terminal fragment within 60 hours of said contacting of said first inhibitor and said second inhibitor, wherein said population of neuroectodermal precursor cells further differentiate into a posterior floor plate tissue or floor plate cells.
 16. A method for inducing differentiation in stem cells, comprising, a) providing: i) a cell culture comprising human pluripotent stem cells, and ii) a first inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling, wherein said first inhibitor is selected from the group consisting of Noggin, a disulfide-linked homodimer of Noggin, Dorsomorphin, LDN-193189, and mixtures thereof, and iii) a second inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling, wherein said second inhibitor is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl] benzamide (SB431542), b) incubating said pluripotent stem cells in a culture medium, c) contacting said pluripotent stem cells within 72 hours of said incubating with said first inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling and said second inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling, and d) inducing differentiation of said contacted pluripotent stem cells into a population of cultured neuroectodermal precursor cells.
 17. The method of claim 16, wherein said culture medium is selected from the group consisting of a knockout replacement medium and an N2 culture medium.
 18. The method of claim 16, wherein said method further comprises a step of removing said first inhibitor from said contacted pluripotent stem cells within 48 hours after contacting said contacted pluripotent stem cells with said first inhibitor wherein said population of cultured neuroectodermal precursor cells comprise placode precursor cells.
 19. The method of claim 12, wherein said placode precursor cells further differentiate into Brn3a+ progenitor cells under serum free conditions.
 20. The method of claim 16, wherein said population of neuroectodermal precursor cells further differentiate into cells selected from the group consisting of central nervous system (CNS) progenitor cells, patternable neuronal cells, dopamine positive neurons and motoneurons. 